Preparation and use of bifunctional molecules having DNA sequence binding specificity

ABSTRACT

Small molecule polyamides that specifically bind with subnanomolar affinity to any predetermined sequence in the human genome with potential use in molecular biology and human medicine are described. Further, the designed compounds which target the minor groove of B-form double helical DNA offer a general approach for the control of gene-expression. Simple rules are disclosed which provide for rational control of the DNA-binding sequence specificity of synthetic polyamides containing N-methylpyrrole and N-methylimidazole amino acids. A series of molecular templates for polyamide design are disclosed which provide for small molecules which recognize predetermined DNA sequences with affinities and specificities comparable to sequence-specific DNA-binding proteins such as transcription factors. These design rule are applied to provide a polyamide for specific targeting of a predetermined 7 base pair sequence from a conserved HIV gene promoter at subnanomolar concentration. The pyrrole-imidazole polyamides described herein represent the only class of designed small molecules to date that can bind any predetermined sequence of double helical DNA.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application Number:PCT/US98/06997, filed on Apr. 8, 1998, entitled DNA-BINDING PYRROLE ANDIMIDAZOLE POLYAMIDE DERIVATIVES, which is a continuation-in-part ofPCT/US97/03332 filed Feb. 20, 1997, Ser. No. 08/853,522 filed May 8,1997 and PCT/US 97/12722 filed Jul. 21, 1997 which arecontinuation-in-part applications of Ser. No. 08/837,524 filed Apr. 21,1997 and Ser. No. 08/607,078 filed Feb. 26, 1996, U.S. provisionalapplication 60/043,444 filed Apr. 8, 1997, U.S. provisional application60/043,446 filed Apr. 8, 1997 and U.S. Provisional application60/042,002 filed Apr. 16, 1997.

This work was supported in part by a grant from the National Institutesof Health (GM-27681). The United States Government may have certainrights to this invention.

FIELD OF THE INVENTION

This invention relates to the fields of molecular biology, biochemistry,and drug design. More particularly, the present invention providessynthetic polyamides containing pyrrole and imidazole amino acids whichbind specific base pair sequences of double helical DNA with affinitiesand specificities comparable to DNA binding proteins such as thetranscription factors. A series of molecular templates are describedwhich allow for rational targeting of any predetermined DNA sequence oftherapeutic potential. This non-biological approach to DNA recognitionprovides an underpinning for the design of synthetic cell-permeableligands for the control of gene-expression.

BACKGROUND OF THE INVENTION

In every human cell, genetic information is stored on a string-like DNApolymer which is approximately 1 meter in length and contains 3×10⁹units of information in the form of base pairs, within which is encodedapproximately 80,000 to 100,000 genes or sets of instructions. (Watson,J. D. Gene, 135, 309-315 (1993).) The specific interaction of proteinssuch as transcription factors with DNA controls the regulation of genesand hence cellular processes. (Roeder, R. G. TIBS, 9, 327-335 (1996).) Awide variety of human conditions ranging from cancer to viral infectionarise from malfunctions in the biochemical machinery that regulatesgene-expression (R. Tijan, Sci. Am., 2, 54-61 (1995).) Designed smallmolecules which target specific DNA sequences offer a potentiallygeneral approach for gene-specific regulation. (Gottesfeld, et al.Nature Accepted. (1997). Such molecules could be powerful therapeuticsfor combating life threatening diseases which result from misregulationin transcription.

Designed bifunctional small molecules which target specific DNAsequences offer a potentially general approach for gene-specific,sequence-specific, or organism specific modification, detection orcapture of plasmids, genes, cDNA, cosmids, or chromosomes. Morespecifically, a life threatening disease may result from a single errorwithin the 3×10⁹ units of information stored within the double helix.Sequence-specific polyamides may discriminate such small errors, hencebifunctional polyamides could have broad diagnostic applications whichrange from determining the molecular basis of life threatening diseasesto sequence-specific visualization of disease genes in living organisms.

The genetic information is in fact, stored on two stands of DNA (inantiparallel orientation) in a structure termed the double helix. TheDNA double helix consists of A,T and G,C base pairs held together byspecific Watson-Crick hydrogen bonds like rungs on a twisted ladder.(Dickerson, et al. Science, 216, 475 (1982). The common B-form of DNA ischaracterized by a wide (12 Å) and shallow major groove and a deep andnarrow (4-6 Å) minor. Individual sequences may be distinguished by thepattern of hydrogen bond donors and acceptors displayed on the edges ofthe base pairs. (Principles of Nucleic Acid Structure Sanger, W.;Springer-Verlag, New York, 1984.) In the minor groove, the A,T base pairpresents two symmetrically placed hydrogen bond acceptors in the minorgroove, the purine N3 and the pyrimidine O2 atoms. The G,C base pairpresents these two acceptors, but in addition presents a hydrogen bonddonor, the 2-amino group of guanine (Steitz, T. A. Quart. Rev. Biophys.23, 205).

Small molecules isolated from natural sources which bind DNA are foundto be a structurally diverse class, as evidenced by consideration offour representative molecules, chromomycin, distamycin, actinomycin D,and calicheamicin. (Gao, et al. J. Mol. Biol. 223, 259-279. (1992);Kamitori, et al. J. Mol. Biol. 225, 445-456 (1992); Paloma et al. J. Am.Chem. Soc. 116, 3697-3708 (1994); Coll, et al. Proc. Natl. Acad. Sci.U.S.A. 84, 8385-8389 (1987)). There is no simple natural recognitioncode for the readout of specific sequences of DNA.

The structures of four small molecules isolated from natural sources areshown in FIG. 1. Among these DNA-binding molecules, distamycin isdistinguished by its structural simplicity, having no chiral centers andan oligopyrrolecarboxamide core structure. (Zimmer, C. Prog. NucleicAcid Res. Mol. Biol. (1975) 15, 285; Baguley, B. C. Molecular andCellular Biochemistry (1982) 43, 167-181; Zimmer, et al., Prog. Biophy.Mol. Biol. 47, 31 (1986)). Structural studies of distamycin-DNAcomplexes reveal modular complexes in which adjacent pyrrolecarboxamidesmakes similar contacts with adjacent DNA base pairs. The relativesimplicity of distamycin, with respect both to its chemical structureand its complexes with DNA, guided the initial decision to usedistamycin as a basis for designed polyamides having novel DNA-bindingsequences specificity. (Dervan, P. B. Science 232, 464-471 (1986).)

A schematic representation of recognition of A,T rich sequences in theminor groove by Distamycin is shown below:

Two distinct DNA binding modes exist for Distamycin A. In the firstbinding mode, a single molecule of Distamycin binds in the middle of theminor groove of a 5 base pair of A,T rich sequence. The amine hydrogensof the N-methylpyrrole-carboxamides form bifurcated hydrogen bonds withAdenine N3 and thymine O2 atoms on the floor of the minor groove.¹⁰ Inthe second binding mode, 2 distamycin ligands form an antiparallelside-by-side dimer in the minor groove of a 5 base pair A,T rich site.(Pelton, J. G. & Wemmer, D. E. (1989) Proc. Natl. Acad. Sci. 86,5723-5727; Pelton, J. G. & Wemmer, D. E. (1990) J. Am. Chem. Soc. 112,1393-1399; Chen, et al. (1994). Nature Struct. Biol. 1, 169-175.) In the2:1 model each polyamide subunit forms hydrogen bonds to a unique DNAstrand in the minor groove.

Polyamides containing N-methylpyrrole (Py) and N-methylimidazole (Im)amino acids provide a model for the design of artificial molecules forrecognition of double helical DNA. For side-by-side complexes ofPy/Im-polyamides in the minor groove of DNA, the DNA binding sequencespecificity depends on the sequence of side-by-side amino acid pairings.(Wade, et al. (1992). J. Am. Chem. Soc. 114, 8783-8794; Mrksich, et al.(1992). Proc. Natl. Acad. Sci. U.S.A. 89, 7586-7590; Wade, W. S.,Mrksich, M. & Dervan, P. B. (1993); Biochemistry 32, 11385-11389(1993)). A pairing of Im opposite Py targets a G•C base pair while apairing of Py opposite Im targets a C•G base pair. A Py/Py combinationis degenerate targeting both A•T and T•A base pairs. Specificity for G,Cbase pairs results from the formation of a putative hydrogen bondbetween the imidazole N3 and the exocyclic amine group of guanine.Validity of the pairing rules is supported by a variety of footprintingand NMR structure studies. (Mrksich, et al., J. Am. Chem. Soc., 115,2572 (1993); Geierstanger, et al. Science, 266, 646 (1994); Mrksich etal., J. Am. Chem. Soc., 117, 3325 (1995).)

A schematic representation of the polyamide pairing rules is shownbelow:

In parallel with the elucidation of the scope and limitations of thepairing rules, efforts have been made to increase the DNA-bindingaffinity and specificity of pyrrole-imidazole polyamides by covalentlylinking polyamide subunits. (Mrksich, M. & Dervan, P. B. (1993). J. Am.Chem. Soc. 115, 9892-9899; Dwyer, et al. (1993). J. Am. Chem. Soc. 115,9900-9906; Mrksich, M. & Dervan, P. B. (1994). J. Am. Chem. Soc. 116,3663-3664; Chen, Y. H. and Lown, J. W. (1994) J. Am. Chem. Soc. 116,6995-7005. Chen, Y. H. and Lown, J. W. Heterocycles 41, 1691-1707(1995). Geierstanger, et al., Nature Structural Biology, 3, 321 (1996).Chen, et al. J. Biomol. Struct. Dyn. 14, 341-355 (1996); Cho, et al.Proc. Natl. Acad. Sci. USA, 92, 10389 (1995)). A simple hairpinpolyamide motif with γ-aminobutyric acid (γ) serving as a turn-specificinternal-guide-residue provides a synthetically accessible method oflinking polyamide subunits within the 2:1 motif. The head-to-tail linkedpolyamide ImPyPy-γ-PyPyPy-dimethylaminopropylamide (Dp) was shown tospecifically bind the designated target site 5′-TGTTA-3′ with anequilibrium association constant of K_(a)=8×10⁷ M⁻¹, an increase of300-fold relative to the unlinked three-ring polyamide pair ImPyPy andPyPyPy. (Mrksich, et al. J. Am. Chem. Soc. 116, 7983-7988). The hairpinpolyamide model is supported by footprinting, affinity cleaving and NMRstructure studies. (Church, et al. Biochemistry 1990, 29, 6827; He, etal. J. Am. Chem. Soc. 1993, 115, 7061; de Clairac, et al. J. Am. Chem.Soc. submitted).

A schematic representation of recognition of a 5′-TGTTA-3′ sequence byunlinked subunits (left) and γ-aminobutyric acid linked subunits (right)is shown below:

Closing the ends of the hairpin to form a cyclic polyamide increases theoverall energetics for DNA-binding presumably by restrictingconformational space for the molecule. (Lown, J. W. and Krowicki, K. J.Org. Chem. 1985, 50, 3774.) A cyclic polyamidecyclo-(ImPyPy-γ-PyPyPy-γ-) was shown to specifically bind the designatedtarget site 5′-TGTTA-3′ with an equilibrium association constant ofK_(a)=2.9×10⁹ M⁻¹, an increase of 40-fold relative to the correspondinghairpin polyamide of sequence composition ImPyPy-γ-PyPyPy. Thesequence-specificity versus single base pair mismatch sites drops from30-fold for the hairpin polyamide to 2-fold for the cyclic polyamide.

A schematic representation of a cyclic polyamide recognizing the minorgroove is shown below:

Despite the design breakthrough in molecular recognition of DNA, thebinding affinities of linked and unlinked polyamide dimers of the priorart are modest when compared to those found with natural DNA bindingproteins. (Clemens, et al. J. Mol. Biol. 244, 23-25 (1994)). For exampleDNA-binding transcription factors recognize their cognate sites atsubnanomolar concentrations. (Jamieson, et al. Biochemistry 33,5689-5695 (1994); Choo, Y. and Klug, A. Proc.Natl. Acad. Sci. U.S.A. 91,11168-11172 (1994); Greisman, H. A. and Pabo, C. O. Science 275, 657-661(1997)). Six-ring hairpin polyamides require concentrations greater than10 nM to occupy their target sites. The only class of polyamidesdescribed in the prior art with affinities similar to DNA-bindingproteins are the 6-ring cyclic polyamides; however, this class ofmolecules lacks the sequence-specificity of proteins (i.e. an energeticpenalty for binding a single base pair mismatch site) and thereforecurrently has no potential for therapeutic applications.

Two prior approaches for the development of synthetic transcriptionalantagonists have been reported. Oligodeoxynucleotides which recognizethe major groove of double helical DNA via triple helix formation bind abroad sequence repertoire with high affinity and specificity (Moser, H.E. & Dervan, P. B. Science 238, 645-650 (1987); Thuong, et al. Agnew.Chem. Int. Ed. Engl. 32, 666-690 (1993)). Although oligonucleotides andtheir analogs have been shown to interfere with gene expression (Maher,et al. Biochemistry 31, 70-81 (1992); Duvalvalentin, et al. Proc. Natl.Acad. Sci. U.S.A. 89, 504-508 (1992)). The triple helix approach islimited to purine tracks and suffers from poor cellular uptake. Thereare a few examples of cell-permeable carbohydrate based ligands thatinterfere with transcription factor function. (Ho, et al. Proc. Natl.Acad. Sci. USA 91, 9203-9207 (1994); Liu, C. et al. Proc. Natl. Acad.Sci. USA 93, 940-944 (1996)). However oligosaccharides are not yetamenable to recognition of a broad range of predetermined DNA sequences.

Because of the small size and hydrophobic nature of polyamides (MW≈1200)and because the parent ligand Distamycin is itself cell-permeable theseligands have the potential to underpin a new field of small moleculeregulation of gene expression. It remained to be determined if lowmolecular weight (MW≈1200) pyrrole-imidazole polyamides could beconstructed which recognize predetermined DNA sites at subnanomolarconcentrations without compromising sequence-selectivity.

SUMMARY OF THE INVENTION

This invention provides improved polyamides for selectively binding aDNA molecule. Compounds of the present invention comprise a polyamide ofthe formula:

where

R¹, R^(a), R^(b), R^(e), R^(f), R^(i), R^(j), R^(n), and R^(o) arechosen independently from H, Cl, NO, N-acetyl, benzyl, C₁₋₆ alkyl, C₁₋₆alkylamine, C₁₋₆ alkyldiamine, C₁₋₆ alkylcarboxylate, C₁₋₆ alkenyl, andC₁₋₆ alkynyl;

R² is selected from the group consisting of H, NH₂, SH, Cl, Br, F,N-acetyl, and N-formyl;

R³, R^(d), R^(l) and R^(q) are selected independently from the groupconsisting of H, NH₂, OH, SH, Br, Cl, F, OMe, CH₂OH, CH₂SH, CH₂NH₂;

R⁴ is —NH(CH₂)₀₋₆NR⁵R⁶ or NH(CH₂)_(r)CO NH(CH₂)₀₋₆NR⁵R⁶ or NHR⁵ orNH(CH₂)_(r)CONHR⁵, where R⁵ and R⁶ are independently chosen from H, Cl,NO, N-acetyl, benzyl, C₁₋₆ alkyl, C₁₋₆ alkylamine, C₁₋₆ alkyldiamine,C₁₋₆ alkylcarboxylate, C₁₋₆ alkenyl, C₁₋₆L, where L groups areindependently chosen from biotin, oligodeoxynucleotide,N-ethylnitrosourea, fluorescein, bromoacetamide, iodoacetamide,DL-α-lipoic acid, acridine, ethyl red, 4-(psoralen-8-xyloxy)-butyrate,tartaric acid, (+)-α-tocopheral, and C₁₋₆ alkynyl, where r is an integerhaving a value ranging from 0 to 6;

X, X^(a), X^(b), X^(e), X^(f), X^(i), X^(j), X^(n), X^(o) are chosenindependently from the group consisting of N, CH, COH, CCH₃, CNH₂, CCl,CF; and

a, b, c, d, e, f, i, j, k, and m are integers chosen independently,having values ranging from 0 to 5;

or a pharmaceutically acceptable salt thereof.

The invention further comprises a polyamide having the formula:

where

R¹, R^(a(i,m)) and R^(b(j,m)) are chosen independently from H, Cl, NO,N-acetyl, benzyl, C₁₋₆ alkyl, C₁₋₆ alkylamine, C₁₋₆ akyldiamine, C₁₋₆alkylcarboxylate, C₁₋₆ alkenyl, and C₁₋₆ alkynyl;

R² is selected from the group consisting of H, NH₂, SH, Cl, Br, F,N-acetyl, and N-formyl;

R^(f(m)) and R^(c(k,m)) are selected independently from the groupconsisting of H, NH₂, OH, SH, Br, Cl, F, OMe, CH₂OH, CH₂SH, CH₂NH₂;

R⁴ is —NH(CH₂)₀₋₆NR⁵R⁶ or NH(CH₂)_(r)CO NH(CH₂)₀₋₆NR⁵R⁶ or NHR⁵ orNH(CH₂)_(r)CONHR⁵, where R⁵ and R⁶ are independently chosen from H, Cl,NO, N-acetyl, benzyl, C₁₋₆ alkenyl, C₁₋₆ alkylamine, C₁₋₆ alkyldiamine,C₁₋₆ alkylcarboxylate, C₁₋₆ alkenyl, C₁₋₆L, where L groups areindependently chosen from biotin, oligodeoxynucleotide,N-ethylnitrosourea, fluorescein, bromoacetamide, iodoacetamide,DL-α-lipoic acid, acridine, ethyl red, 4-(psoralen-8-yloxy)-butyrate,tartaric acid, (+)-α-tocopheral, and C₁₋₆ alkynyl, where r is an integerhaving a value ranging from 0 to 6;

X, X^(a(i,m)) and X^(b(j,m)) are chosen independently from the groupconsisting of N, CH, COH, CCH₃, CNH₂, CCl, CF; and

a, b, c, d, e, f, g, h, i, j, k, l, m, n, o and p are integers chosenindependently, having values ranging from 0 to 5;

or a pharmaceutically acceptable salt thereof.

By “alkyl” or “lower alkyl” in the present invention is meant C₁-C₆alkyl, i.e., straight or branched chain alkyl groups having 1-6 carbonatoms, such as, for example, methyl, ethyl, propyl, isopropyl, n-butyl,sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl,2-hexyl, 3-hexyl, and 3-methylpentyl. Preferred C₁-C₆ alkyl groups aremethyl, ethyl, propyl, butyl, cyclopropyl or cycloproylmethyl.Particularly preferred are C₁-C alkyl groups such as methyl, ethyl, andpropyl.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Small molecules isolated from natural sources.

FIG. 2. Hairpin polyamides.

FIG. 3. Chemical structures of polyamides.

FIG. 4. Solid phase synthesis of polyamides.

FIG. 5. Extended hairpin polyamides.

FIG. 6. Association profile of extended hairpin polyamides.

FIG. 7. Binding models for polyamides.

FIG. 8. Schematic binding models for eight ring hairpin polyamide.

FIG. 9. Eight-residue hairpin polyamides.

FIG. 10. Structure of 4-β-4 polyamides.

FIG. 11. Recognition of DNA by 4-β-4 polyamides

FIG. 12. Placement of β/β pairs.

FIG. 13. β-linked fully overlapped polyamide complexes.

FIG. 14. 10-ring hairpin polyamides.

FIG. 15. Discrimination of seven base pair sequence by polyamides.

FIG. 16. Hairpin polyamides that recognize seven base pair sequence.

FIG. 17. Dnase I footprint titration.

FIG. 18. Ni(II)-Gly-Gly-His modified polyamide.

FIG. 19. Bromoacetylated hairpin polyamide.

FIG. 20. Structure of (+) CC-1065 and duocarmycins.

FIG. 21. Alkylation mechanism of CC-1065.

FIG. 22. Structure of Bizlesin and CBI.

FIG. 23. Synthesis of CBI-polyamide conjugate.

FIG. 24. Synthesis of bifunctional methidium-polyamide conjugates.

FIG. 25. Synthesis of polyamide-rhodamine conjugate.

FIG. 26. Structure of polyamide-DYE conjugates.

FIG. 27. Synthesis of biotin-polyamide conjugates.

FIG. 28. Bifunctional biotin-polyamide conjugates.

FIG. 29. Affinity capture using bifunctional biotin-polyamideconjugates.

FIG. 30. Psoralen-polyamide conjugate.

FIG. 31. Cooperative dimerization of polyamides.

FIG. 32. Binding of polyamides to mismatched sites.

FIG. 33. Footprint titration of polyamides.

FIG. 34. Generalizable polyamide motifs.

FIG. 35. Examples of polyamides.

FIG. 36. Determination of polyamide affinity.

FIG. 37. N-terminally extended polyamides.

FIG. 38. Polyamides binding 16 base pair sequence.

FIG. 39. Determination of 16 base pair sequence.

FIG. 40. Binding of polyamides to mismatched sites.

FIG. 41. β-substitution in polyamides.

FIG. 42. Affinity determinations for β-substituted polyamides.

FIG. 43. Binding of polyamides to TATA box.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Within this application, unless otherwise stated, definitions of theterms and illustration of the techniques of this application may befound in any of several well-known references such as: Sambrook, J., etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (1989); Goeddel, D., ed., Gene Expression Technology,Methods in Enzymology, 185, Academic Press, San Diego, Calif. (1991);“Guide to Protein Purification” in Deutshcer, M. P., ed., Methods inEnzymology, Academic Press, San Diego, Calif. (1989); Innis, et al., PCRProtocols: A Guide to Methods and Applications, Academic Press, SanDiego, Calif. (1990); Freshney, R. I., Culture of Animal Cells: A Manualof Basic Technique, 2^(nd) Ed., Alan Liss, Inc. New York, N.Y. (1987);Murray, E. J., ed., Gene Transfer and Expression Protocols, pp. 109-128,The Human Press Inc., Clifton, N.J. and Lewin, B., Genes VI, OxfordUniversity Press, New York (1987).

For the purposes of this application, a promoter is a regulatorysequence of DNA that is involved in the binding of RNA polymerase toinitiated transcription of a gene. A gene is a segment of DNA involvedin producing a peptide, polypeptide or protein, including the codingregion, non-coding regions preceding (“leader”) and following(“trailer”) the coding region, as well as intervening non-codingsequences (“introns”) between individual coding segments (“exons”).Coding refers to the representation of amino acids, start and stopsignals in a three base “triplet” code. Promoters are often upstream(“'5 to”) the transcription initiation site of the corresponding gene.Other regulatory sequences of DNA in addition to promoters are known,including sequences involved with the binding of transcription factors,including response elements that are the DNA sequences bound byinducible factors. Enhancers comprise yet another group of regulatorysequences of DNA that can increase the utilization of promoters, and canfunction in either orientation (5′-3′ or 3′-5′) and in any location(upstream or downstream) relative to the promoter. Preferably, theregulatory sequence has a positive activity, i.e., binding of anendogenous ligand (e.g. a transcription factor) to the regulatorysequence increases transcription, thereby resulting in increasedexpression of the corresponding target gene. In such a case,interference with transcription by binding a polyamide to a regulatorysequence would reduce or abolish expression of a gene.

The promoter may also include or be adjacent to a regulatory sequenceknown in the art as a silencer. A silencer sequence generally has anegative regulatory effect on expression of the gene. In such a case,expression of a gene may be increased directly by using a polyamide toprevent binding of a factor to a silencer regulatory sequence orindirectly, by using a polyamide to block transcription of a factor to asilencer regulatory sequence.

It is to be understood that the polyamides of this invention bind todouble stranded DNA in a sequence specific manner. The function of asegment of DNA of a given sequence, such as 5′-TATAAA-3′, depends on itsposition relative to other functional regions in the DNA sequence. Inthis case, if the sequence 5′-TATAAA-3′ on the coding strand of DNA ispositioned about 30 base pairs upstream of the transcription start site,the sequence forms part of the promoter region (Lewin, Genes VI, pp.831-835). On the other hand, if the sequence 5′-TATAAA-3′ is downstreamof the transcription start site in a coding region and in properregister with the reading frame, the sequence encodes the tyrosyl andlysyl amino acid residues (Lewin, Genes VI, pp. 213-215).

While not being held to one hypothesis, it is believed that the bindingof the polyamides of this invention modulate gene expression by alteringthe binding of DNA binding proteins, such as RNA polymerase,transcription factors, TBF, TFIIIB and other proteins. The effect ongene expression of polyamide binding to a segment of double stranded DNAis believed to be related to the function, e.g., promoter, of thatsegment of DNA.

It is to be understood by one skilled in the art that the improvedpolyamides of the present invention may bind to any of theabove-described DNA sequences or any other sequence having a desiredeffect upon expression of a gene. In addition, U.S. Pat. No. 5,578,444describes numerous promoter targeting sequences from which base pairsequences for targeting an improved polyamide of the present inventionmay be identified.

It is generally understood by those skilled in the art that the basicstructure of DNA in a living cell includes both major and a minorgroove. For the purposes of describing the present invention, the minorgroove is the narrow groove of DNA as illustrated in common molecularbiology reference such as Lewin, B., Genes VI, Oxford University Press,New York (1997).

To affect gene expression in a cell, which may include causing anincrease or a decrease in gene expression, a effective quantity of oneor more polyamide is contacted with the cell and internalized by thecell. The cell may be contacted in vivo or in vitro. Effectiveextracellular concentrations of polyamides that can modulate geneexpression range from about 10 nanomolar to about 1 micromolar.Gottesfeld, J. M., et al., Nature 387 202-205 (1997). To determineeffective amounts and concentrations of polyamides in vitro, a suitablenumber of cells is plated on tissue culture plates and variousquantities of one or more polyamide are added to separate wells. Geneexpression following exposure to a polyamide can be monitored in thecells or medium by detecting the amount of the protein gene productpresent as determined by various techniques utilizing specificantibodies, including ELISA and wester blot. Alternatively, geneexpression following exposure to a polyamide can be monitored bydetecting the amount of messenger RNA present as determined by varioustechniques, including northern blot and RT-PCR.

Similarly, to determine effective amounts and concentrations ofpolyamides for in vivo administration, a sample of body tissue or fluid,such as plasma, blood, urine, cerebrospinal fluid, saliva, or biopsy ofskin, muscle, liver, brain or other appropriate tissue source isanalyzed. Gene expression following exposure to a polyamide can bemonitored by detecting the amount of the protein gene product present asdetermined by various techniques utilizing specific antibodies,including ELISA and wester blot. Alternatively, gene expressionfollowing exposure to a polyamide can be monitored by the detecting theamount of messenger RNA present as determined by various techniques,including northern blot and RT-PCR.

The polyamides of this invention may be formulated into diagnostic andtherapeutic compositions for in vivo or in vitro use. Representativemethods of formulation may be found in Remington: The Science andPractice of Pharmacy, 19th ed., Mack Publishing Co., Easton, Pa. (1995).

For in vivo use, the polyamides may be incorporated into aphysiologically acceptable pharmaceutical composition that isadministered to a patient in need of treatment or an animal for medicalor research purposes. The polyamide composition comprisespharmaceutically acceptable carriers, excipients, adjuvants,stabilizers, and vehicles. The composition may be in solid, liquid, gel,or aerosol form. The polyamide composition of the present invention maybe administered in various dosage forms orally, parentally, byinhalation spray, rectally, or topically. The term parenteral as usedherein includes, subcutaneous, intravenous, intramuscular, intrasternal,infusion techniques or intraperitoneally.

The selection of the precise concentration, composition, and deliveryregimen is influenced by, inter alia, the specific pharmacologicalproperties of the particular selected compound, the intended use, thenature and severity of the condition being treated or diagnosed, theage, weight, gender, physical condition and mental acuity of theintended recipient as well as the route of administration. Suchconsiderations are within the purview of the skilled artisan. Thus, thedosage regimen may vary widely, but can be determined routinely usingstandard methods.

Polyamides of the present invention are also useful for detecting thepresence of double stranded DNA of a specific sequence for diagnostic orpreparative purposes. The sample containing the double stranded DNA canbe contacted by polyamide linked to a solid substrate, thereby isolatingDNA comprising a desired sequence. Alternatively, polyamides linked to asuitable detectable marker, such as biotin, a hapten, a radioisotope ora dye molecule, can be contacted by a sample containing double strandedDNA.

The design of bifunctional sequence specific DNA binding moleculesrequires the integration of two separate entities: recognition andfunctional activity. Polyamides that specifically bind with subnanomolaraffinity to the minor groove of a predetermined sequence of doublestranded DNA are linked to a functional molecule, providing thecorresponding bifunctional conjugates useful in molecular biology,genomic sequencing, and human medicine. Polyamides of this invention canbe conjugated to a variety of functional molecules, which can beindependently chosen from but is not limited to arylboronic acids,biotins, polyhistidines comprised from about 2 to 8 amino acids, haptensto which an antibody binds, solid phase supports, oligodeoxynucleotides,N-ethylnitrosourea, fluorescein, bromoacetamide, iodoacetamide,DL-α-lipoic acid, acridine, captothesin, pyrene, mitomycin, texas red,anthracene, anthrinilic acid, avidin, DAPI, isosulfan blue, malachitegreen, psoralen, ethyl red, 4-(psoraen-8-yloxy)-butyrate, tartaric acid,(+)-α-tocopheral, psoralen, EDTA, methidium, acridine,Ni(II)•Gly-Gly-His, TO, Dansyl, pyrene, N-bromoacetamide, and goldparticles. Such bifunctional polyamides are useful for DNA affinitycapture, covalent DNA modification, oxidative DNA cleavage, DNAphotocleavage. Such bifunctional polyamides are useful for DNA detectionby providing a polyamide linked to a detectable label. DNA complexed toa labeled polyamide can then be determined using the appropriatedetection system as is well known to one skilled in the art. Forexample, DNA associated with a polyamide linked to biotin can bedetected by a streptavidin/alkaline phosphatase system.

The present invention also describes a diagnostic system, preferably inkit form, for assaying for the presence of the double stranded DNAsequence bound by the polyamide of this invention in a body sample, suchbrain tissue, cell suspensions or tissue sections, or body fluid samplessuch as CSF, blood, plasma or serum, where it is desirable to detect thepresence, and preferably the amount, of the double stranded DNA sequencebound by the polyamide in the sample according to the diagnostic methodsdescribed herein.

The diagnostic system includes, in an amount sufficient to perform atleast one assay, a specific polyamide as a separately packaged reagent.Instructions for use of the packaged reagent(s) are also typicallyincluded. As used herein, the term “package” refers to a solid matrix ormaterial such as glass, plastic (e.g., polyethylene, polypropylene orpolycarbonate), paper, foil and the like capable of holding within fixedlimits a polyamide of the present invention. Thus, for example, apackage can be a glass vial used to contain milligram quantities of acontemplated polyamide or it can be a microliter plate well to whichmicrogram quantities of a contemplated polyamide have been operativelyaffixed, i.e., linked so as to be capable of being bound by the targetDNA sequence. “Instructions for use” typically include a tangibleexpression describing the reagent concentration or at least one assaymethod parameter such as the relative amounts of reagent and sample tobe admixed, maintenance time periods for reagent or sample admixtures,temperature, buffer conditions and the like. A diagnostic system of thepresent invention preferably also includes a detectable label and adetecting or indicating means capable of signaling the binding of thecontemplated polyamide of the present invention to the target DNAsequence. As noted above, numerous detectable labels, such as biotin,and detecting or indicating means, such as enzyme-linked (direct orindirect) streptavidin, are well known in the art.

Trauger, et al. (Nature, 382: 559-561) and Swalley, et al. (J. Am. Chem.Soc. 119: 6953-6961) have described recognition of DNA by certainpolyamides at subnanomolar concentrations. Pairing specific carboxyamidegroups allows for recognition of specific DNA sequences (Swalley, et al.supra). Polyamides comprising Hp, Im, and Py provide for coded targetingof pre-determined DNA sequences with high affinity and specificity. Imand Py polyamides may be combined to form Im/Py, Py/Im, Py/Py bindingpairs which complement the four Watson-Crick base pairs A, C, G, and T.Table 1 illustrates such pairings.

TABLE 1 Pairing Codes for Base Pair Recognition* Pair G · C C · G T · AA · T Im/Py + − − − Py/Im − + − − Im/β + − − − β /Im − + − − Py/Py −− + + *favored (+), disfavored (−)

The basic polyamide pairing rules of the prior art are insufficient fordesign of ligands recognizing target sites having subnanomolar bindingaffinities. Additional second generation rules for polyamide design areprovided herein. Each additional rule alone may not be sufficient fordesign of polyamides with subnanomolar affinity. However, simultaneousapplication of the second generation design rules provided herein allowsfor the construction of a number of versatile molecular templates forpolyamide design.

It has been found that a hairpin polyamide synthesized fromBoc-β-alanine-Pam-Resin, ImPyPy-γ-PyPyPy-β-Dp binds with both enhancedaffinity and specificity relative to the parent compound,ImPyPy-γ-PyPyPy-Dp, which lacks the C-terminal β-alanine residue.(Optimization of the Hairpin Polyamide Design for Recognition of theMinor Groove of DNA. M. E. Parks, E. E. Baird and P. B. Dervan, J. Am.Chem. Soc., 118, 6147 (1996).) More specifically ImPyPy-γ-PyPyPy-β-Dpbinds with an apparent first order association constant, K_(a)=3×10⁸M⁻¹, a factor of four greater than the parent polyamide,ImPyPy-γ-PyPyPy-Dp, K_(a)=8×10⁷ M⁻¹. Furthermore, ImPyPy-γ-PyPyPy-β-Dpbinds the target 5′-TGTTA-3′ match site with 60-fold specificityrelative to a single base pair 5′-TGACA-3′ mismatch site. This can becompared with the parent polyamide ImPyPy-γ-PyPyPy-Dp which has a24-fold specific binding relative to the same two DNA sites. The modestincreased binding affinity of the C-terminal β-alanine polyamide, mayresult from an additional hydrogen bond between the β-alaninecarboxamide and a ‘sixth’ base pair of the binding site.

Three or four-ring improved polyamides of the present invention arecovalently coupled to form six or eight-ring structures, respectively,that bind specifically to four or six base pair targets, respectively,at subnanomolar concentrations. As such, the improved polyamides of thepresent invention may be directed to any DNA sequence comprised of A, C,G, or T.

In one embodiment, the present invention comprises improved polyamideshaving three or four-ring polyamide structures covalently coupled toform six or eight-ring hairpin structures, respectively, of the generalstructures I-XXVIII:

X₁X₂X₃X₄γX₅X₆X₇X₈ X₁X₂X₃X₄X₅γX₆X₇X₈X₉X₁₀ I II X₁X₂X₃βX₄X₅X₆X₁X₂X₃X₄βX₅X₆X₇X₈ III IV X₁X₂X₃X₄X₅βX₆X₇X₈ X₁X₂X₃X₄βX₅X₆X₇ V VIX₁X₂X₃βX₄X₅X₆X₇X₈ X₁X₂X₃X₄βX₅X₆X₇X₈ VII VIII X₁X₂X₃X₄X₅βX₆X₇X₈X₉X₁₀X₁X₂X₃X₄X₅X₆γX₇X₈X₉X₁₀X₁₁X₁₂ IX X X₁X₂X₃X₄X₅βγX₆X₇X₈X₉X₁₀X₁βX₂X₃γX₄βX₅X₆ XI XII X₁X₂βX₃X₄γX₅X₆βX₇X₈ X₁X₂βX₃X₄X₅γX₆X₇X₈βX₉X₁₀ XIIXIV X₁X₂X₃βX₄X₅γX₆X₇X₈βX₉X₁₀ X₁X₂X₃X₄βX₅γX₆βX₇X₈X₉X₁₀ XV XVIX₁βX₂X₃X₄X₅γX₆βX₇X₈X₉X₁₀ X₁X₂X₃X₄βX₅γX₆X₇X₈X₉βX₁₀ XVII XVIIIX₁X₂X₃βX₄X₅γX₆X₇βX₈X₉X₁₀ X₁X₂βX₃X₄X₅γX₆X₇βX₈X₉X₁₀ XIX XXX₁βX₂X₃X₄X₅γX₆X₇X₈X₉βX₁₀ X₁X₂βX₃X₄βX₅X₆βX₇X₈ XXI XXIIX₁X₂X₃βX₄X₅X₆βX₇X₈X₉ X₁X₂X₃X₄βX₅βX₆X₇X₈X₉ XXIII XXIVX₁X₂X₃γX₄X₅X₆βX₇X₈X₉ X₁X₂X₃γX₄X₅X₆βX₇X₈X₉βX₁₀X₁₁X₁₂ XXV XXVIX₁X₂X₃γX₄X₅X₆GX₇X₈X₉ X₁X₂X₃X₄γX₅X₆X₇X₈βX₉X₁₀X₁₁X₁₂ XXVII XXVIII

where X₁₋₁₂ is a substituted imidazole such asN-methylimidazolecarboxamide (Im), or a substituted pyrrole such asN-methylpyrrolecarboxamide (Py). An improved polyamide of the presentinvention may also include a C-terminal aliphatic amino acid such as aβ-alanine residue (β) joined to an amide group such asdimethylaminopropylamide (Dp). In addition, an improved polyamide of thepresent invention may further include a aliphatic amino acid such asβ-alanine residue (β) or glycine (G), an amide group such asdimethylaminopropylamide (Dp), an alcohol such as EtOH, an acid such asethylenediaminetetraacetic acid (EDTA), or any derivative thereof joinedto the γ-aminobutyric acid (γ) residue.

The use of β-alanine in the synthetic methods providesaromatic/aliphatic pairing (Im/β, β/Im, Py/β, and β/Py) andaliphatic/aliphatic pairing (β/β) substitution. The use ofγ-aminobutyric acid, or a substituted γ-aminobutyric acid such as(R)-2,4 diaminobutyric acid, provides for preferred hairpin turns. Manyother groups suitable for the purposes of practicing this invention arewell known and widely available to one skilled in the art.

The polyamide subunit structures I-XXVIII above, and XXIX below may becovalently coupled through the γ residue which represents a—NH—CH₂—CH₂—CH₂—CONH— hairpin linkage derived from γ-aminobutyric acidor a chiral hairpin linkage derived from R-2,4-diaminobutyric acid. Thepresent invention provides the reagents and methodologies forsubstituting the γ-residue of certain polyamides with a moiety such as(R)-2,4,-diaminobutyric acid ((R)^(H) ^(₂) ^(N)γ). The NMR structure ofa hairpin polyamide of sequence composition ImPyPy-γ-PyPyPy complexedwith a 5′-TGTTA-3′ target site indicated that it was possible tosubstitute the α-position of the γ-aminobutyric acid residue within thehairpin-DNA complex (de Claire, et al. J. Am. Chem. Soc. 1997, 119,7909). Modeling indicated that replacing the α-H of γ with an aminogroup that may confer an R-configuration at the α-carbon and could beaccommodated within the floor and walls of the minor groove.

A polyamide of Formulas I-XXIX may also be conjugated to a bifunctionalgroup including but not limited to arylboronic acid, biotins,polyhistidine of 2 to 8 amino acids, hapten to which an antibody binds,solid phase support, oligodeoxynucleotide, N-ethylnitrosourea,fluorescein, bromoacetamide, iodoacetamide, DL-α-lipoic acid, acridine,captothesin, pyrene, mitomycin, texas red, anthracene, anthrinilic acid,avidin, DAPI, isosulfan blue, malachite green, psoralen, ethyl red,4-(psoraen-8-yloxy)-butyrate, tartaric acid, or (+)-α-tocopheral. Manyother groups suitable for the purposes of practicing this invention arewell known and widely available to one skilled in the art.

As used herein “polyamide” refers to a polymer comprising the subunitslisted below:

where

R¹ is C₁₋₁₀₀ alkyl (preferably C₁₋₁₀ alkyl such as methyl, ethyl,isopropyl), C₁₋₁₀₀ alkylamine (preferably C₁₋₁₀ alkylamine such asethylamine), C₁₋₁₀₀ alkyldiamine (preferably C₁₋₁₀ alkyldiamine such asN,N-dimethylpropylamine), C₁₋₁₀₀ alkylcarboxylate (preferably a C₁₋₁₀alkylcarboxylate such as —CH₂COOH), C₁₋₁₀₀ alkenyl (preferably C₁₋₁₀alkenyl such as CH₂CH═CH₂), C₁₋₁₀₀ alkynyl (preferably C₁₋₁₀ alkynylsuch as CH₂C≡CH₃), or C₁₋₁₀₀L;

L includes but is not limited to an arylboronic acid, biotin,polyhistidine comprising from 2 to 8 amino acids, hapten to which anantibody binds, solid phase support, oligodeoxynucleotide,N-ethylnitrosourea, fluorescein, bromoacetamide, iodoacetamide,DL-α-lipoic acid, acridine, captothesin, pyrene, mitomycin, texas red,anthracene, anthrinilic acid, avidin, DAPI, isosulfan blue, malachitegreen, psoralen, ethyl red, 4-(psoralen-8-yloxy)-butyrate, tartaricacid, and (+)-α-tocopheral;

m is an integer value ranging from 0 to 12;

R² is H, NH₂, SH, Cl, Br, F, N-acetyl, or N-formyl;

R3 is H, NH₂, OH, SH, Br, Cl, F, OMe, CH₂OH, CH₂SH, or CH₂NH₂; and,

X is N, CH, COH, CCH₃, CNH₃, CCl, or CF.

In a preferred embodiment, R⁵ and R⁶ are H.

The compounds of the present invention may comprise a compound ofFormula XXIX or XXX:

where

R¹, R^(a), R^(b), R^(e), R^(f), R^(i), R^(j), R^(n), and R^(o) arechosen independently from H, Cl, NO, N-acetyl, benzyl, C₁₋₆ alkyl, C₁₋₆alkylamine, C₁₋₆ alkyldiamine, C₁₋₆ alkylcarboxylate, C₁₋₆ alkenyl, andC₁₋₆ alkynyl;

R² is selected from the group consisting of H, NH₂, SH, Cl, Br, F,N-acetyl, and N-formyl;

R³, R^(d), R¹ and R^(q) are selected independently from the groupconsisting of H, NH₂, OH, SH, Br, Cl, F, OMe, CH₂OH, CH₂SH, CH₂NH₂;

R⁴ is —NH(CH₂)₀₋₆NR⁵R⁶ or NH(CH₂)_(r)CO NH(CH₂)₀₋₆NR⁵R⁶ or NHR⁵ orNH(CH₂)_(r)CONHR⁵, where R⁵ and R⁶ are independently chosen from H, Cl,NO, N-acetyl, benzyl, C₁₋₆ alkyl, C₁₋₆ alkylamine, C₁₋₆ alkyldiamine,C₁₋₆ alkylcarboxylate, C₁₋₆ alkenyl, C₁₋₆L, where L groups areindependently chosen from biotin, oligodeoxynucleotide,N-ethylnitrosourea, fluorescein, bromoacetamide, iodoacetamide,DL-α-lipoic acid, acridine, ethyl red, 4-(psoralen-8-yloxy)-butyrate,tartaric acid, (+)-α-tocopheral, and C₁₋₆ alkynyl, where r is an integerhaving a value ranging from 0 to 6;

X, X^(a), X^(b), X^(e), X^(f), X^(i), X^(j), X^(n), X^(o) are chosenindependently from the group consisting of N, CH, COH, CCH₃, CNH₂, CCl,CF; and

a, b, c, d, e, f, i, j, k, and m are integers chosen independently,having values ranging from 0 to 5;

or a pharmaceutically acceptable salt thereof.

The invention further comprises a polyamide having the formula:

where

R¹, R^(a(i,m)) and R^(b(j,m)) are chosen independently from H, Cl, NO,N-acetyl, benzyl, C₁₋₆ alkyl, C₁₋₆ alkylamine, C₁₋₆ alkyldiamine, C₁₋₆alkylcarboxylate, C₁₋₆ alkenyl, and C₁₋₆ alkynyl;

R² is selected from the group consisting of H, NH₂, SH, Cl, Br, F,N-acetyl, and N-formyl;

R^(f(m)) and R^(c(k,m)) are selected independently from the groupconsisting of H, NH₂, OH, SH, Br, Cl, F, OMe, CH₂OH, CH₂SH, CH₂NH₂;

R⁴ is —NH(CH₂)₀₋₆NR⁵R⁶ or NH(CH₂)_(r)CO NH(CH₂)₀₋₆NR⁵R⁶ or NHR⁵ orNH(CH₂)_(r)CONHR⁵, where R⁵ and R⁶ are independently chosen from H, Cl,NO, N-acetyl, benzyl, C₁₋₆ alkyl, C₁₋₆ alkylamine, C₁₋₆ alkyldiamine,C₁₋₆ alkylcarboxylate, C₁₋₆ alkenyl, C₁₋₆L, where L groups areindependently chosen from biotin, oligodeoxynucleotide,N-ethylnitrosourea, fluorescein, bromoacetamide, iodoacetamide,DL-α-lipoic acid, acridine, ethyl red, 4-(psoralen-8-yloxy)-butyrate,tartaric acid, (+)-α-tocopheral, and C₁₋₆ alkynyl, where r is an integerhaving a value ranging from 0 to 6;

X, X^(a(i,m)) and X^(b(j,m)) are chosen independently from the groupconsisting of N, CH, COH, CCH₃, CNH₂, CCl, CF; and

a, b, c, d, e, f, g, h, i, j, k, l, m, n, o and p are integers chosenindependently, having values ranging from 0 to 5;

or a pharmaceutically acceptable salt thereof.

Baird, et al. (J. Am. Chem. Soc. 118: 6141-6146) and PCT/US97/003332describe methods for synthesis of polyamides which are suitable forpreparing polyamides of this invention. Polyamides of the presentinvention may be synthesized by solid phase methods using compounds suchas Boc-protected 3-methoxypyrrole, imidazole, and pyrrole aromatic aminoacids, which are cleaved from the support by aminolysis, deprotectedwith sodium thiophenoxide, and purified by reverse-phase HPLC. Theidentity and purity of the polyamides may be verified using any of avariety of analytical techniques available to one skilled in the artsuch as 1H-NMR, analytical HPLC, and/or matrix-assisted laser-desorptionionization time-of-flight mass spectrometry (MALDI-TOFMS-monoisotropic).

In addition, the above polyamide subunits can be synthesized in smallscale by methods known in the art. The synthesis of Boc-Py-OBt ester 7(Grehn, L. and Ragnarsson, U. J. Org. Chem. 1981, 46, 3492.) and Boc-Imacid 11 (Grehn, et al. Acta. Chim. Scand. 1990, 44, 67.) has beenpreviously described. Available procedures provide only milligram togram quantities of monomer (J. Org. Chem. 52, 3493-3500 (1987); Bailey,et al. Org. Synth. 51, 101 (1971); Nishsiwaki, et al. Heterocycles 27,1945 (1988). Bailey, et al. J. Pharm. Sci. 78, 910, (1989)), whilerequiring difficult column chromatography and the use of toxicchlorofluorophosgene for introduction of the Boc group. An optimizedsynthesis, using inexpensive starting materials, has been developed bythe present inventor allowing Boc-Py-OBt ester and Boc-Im acid monomersto be prepared on 50 g scale without the use of column chromatography.Two dimeric building blocks have also been prepared, Boc-Py-Im acid andBoc-γ-Im acid.

A general method for preparation of these compounds is as follows:

The polyamide polymer can be a homopolymer of Py and Im subunits or acopolymer with strategically placed aliphatic amino acid monomers suchas α-amino acids (including but not limited to the naturally occurringamino acids and preferably being glycine); amino acids of the formula—NH—(CH)_(n)—CO—, where n is an integer from 1-12 (preferably n being 1as in β-alanine or 2 as in γ-aminobutyric acid).

The carboxy terminus of the polyamide may comprise —NH(CH₂)₀₋₆, NR¹R² orNH(CH₂)_(b)CO NH(CH₂)₀₋₆NR¹R^(2′), NHR¹ or NH(CH₂)_(b)CO NHR¹ where b isan integer from 1-6 (preferably 1) and R¹ and R² are independentlychosen from C₁₋₆ alkyl (preferably C₁₋₃ alkyl such as methyl, ethyl,isopropyl), C₁₋₆ alkylamine (preferably C₁₋₃ alkylamine such asethylamine), C₁₋₆ alkyldiamine (preferably C₁₋₃ alkyldiamine such asN,N-dimethylpropylamine), C₁₋₆ alkylcarboxylate (preferably a C₁₋₃alkylcarboxylate such as —CH₂COOH), C₁₋₆ alkenyl (preferably C₁₋₃alkenyl such as CH₂CH═CH₂), C₁₋₆ alkynyl (preferably C₁₋₃ alkynyl suchas —CH₂C≡CH₃), or a C₁₋₆L where L includes but is not limited to biotin,oligodeoxynucleotide, N-ethylnitrosourea, fluorescein, bromoacetamide,iodoacetamide, DL-α-lipoic acid, acridine, ethyl red,4-(psoraen-8-yloxy)-butyrate, tartaric acid, (+)-α-tocopheral.

Most preferred compounds of the instant invention are polyamides coresequence composition: ImPyPyPy-γ-PyPyPyPy, PyPyImPy-γ-PyPyPyPy,ImPyPyPy-γ-ImPyPyPy, PyImPyPy-γ-PyImPyPy, ImPyImPy-γ-PyPyPyPy,ImImPyPy-γ-PyPyPyPy, ImImImPy-γ-PyPyPyPy, ImImPyPy-γ-ImPyPyPy,ImPyPyPy-γ-ImImPyPy, ImImPyPy-γ-ImImPyPy, ImPyImPy-γ-ImPyImPy,ImImImPy-γ-ImPyPyPyPy, ImImImIm-γ-PyPyPyPy, Im-β-PyPy-γ-Im-β-PyPy,Im-β-ImIm-γ-Py-β-PyPy, Im-β-ImPy-γ-Im-β-ImPy, ImPyPyPyPy-γ-ImPyPyPyPy,ImImPyPyPy-γ-ImPyPyPyPy, ImPyImPyPy-γImPyPyPyPy,ImImPyImIm-γ-PyPyPyPyPy, ImPyPyImPy-γ-ImPyPyImPy,ImPy-β-PyPy-γ-ImPy-β-PyPy, ImIm-β-ImIm-γ-PyPy-β-PyPy,ImPy-β-ImPy-γ-ImPy-β-ImPy ImPy-β-PyPyPy-γ-ImPyPy-β-PyPy,ImIm-β-PyPyPy-γ-PyPyPy-β-PyPy, ImPy-β-ImPyPy-γ-ImPyPy-β-PyPy,ImIm-β-PyPyPy-γ-ImImPy-β-PyPy, ImPy-β-PyPyPy-γ-PyPyPy-β-ImPy,ImPyPyPyPyPy-γ-ImPyPyPyPyPy, ImPyPy-β-PyPy-γ-ImPyPy-β-PyPy,ImPyPyPy-β-Py-γ-Im-β-PyPyPyPy, ImImPyPyPyPy-γ-ImImPyPyPyPy,Im-β-PyPyPyPy-γ-Im-β-PyPyPyPy, ImPyPyPy-β-Py-γ-ImPyPyPy-β-Py,ImPyImPyPyPy-γ-ImPyPyPyPyPy, ImPyPy-β-PyPy-γ-ImPy-β-PyPyPy,ImPyPyPyPy-β-γ-ImPyPyPyPy-β, ImPy-β-ImPyPy-γ-ImPy-β-ImPyPy,Im-β-PyPyPyPy-γ-ImPyPyPy-β-Py, Im-β-ImPyPyPy-γ-ImPyPyPy-β-Py,ImPyPy-β-PyPyPy, ImImPy-β-PyPyPy, ImImIm-β-PyPyPy, ImPyPyPyPy-β-PyPyPy,ImPyPyPy-β-PyPyPy, ImPyPy-β-PyPyPyPyPy, ImPyPyPy-β-PyPyPyPy,ImImPyPy-β-PyPyPyPy, ImImImPy-β-PyPyPyPy, ImPyPyPy-β-ImPyPyPy,ImImPyPy-β-ImPyPyPy, ImImPyPyPy-β-PyPyPyPyPy, ImImImPyPy-β-PyPyPyPyPy,ImIm-β-PyPy-β-PyPy-β-PyPy, ImImPy-β-PyPyPy-β-PyPyPy,ImImPyPy-β-Py-β-PyPyPyPy, ImPyPy-γ-ImPyPy-β-PyPyPy,ImPyPy-γ-PyPyPy-β-PyPyPy, PyImPy-γ-ImPyPy-β-PyPyPy,PyImPy-γ-ImPyPy-β-PyPyPy-β-PyPyPy, ImImPy-γ-ImPyPy-β-PyPyPy,ImPyPy-γ-ImPyPy-G-PyPyPy, ImPyPyPy-γ-ImImImPy-β-PyPyPyPy,ImImPyPy-γ-ImImPyPy-β-PyPyPyPy, and ImImPyPy-γ-PyPyPyPy-β-PyPyPyPy.

The compounds of the following invention may be synthesized by any ofseveral well-known and widely available techniques. Distamycin and itsanalogs can be produced by traditional multi-step synthetic organicchemistry (Weiss, et al. J. Am. Chem. Soc. 1957, 79, 1266; Arcamone, etal. Gazz. Chim. Ital. 1967, 97, 1097; Penco, et al Gazz. Chim. Ital.1967, 97, 1110; Bailer, et al. Tetrahedron 1978, 34, 2389.) Therepeating amide of distamycin is formed from an aromatic carboxylic acidand an aromatic amine, both of which have proven problematic forsolution phase coupling reactions. The aromatic acid is often unstableto decarboxylation and the aromatic amines have been found to be air andlight sensitive. (Lown, et al. J. Org. Chem. 1985, 50, 3774.) Variablecoupling yields, long reaction times (often>24 h); numerous sideproducts, and reactive intermediates (acid chlorides andtrichloroketones) are characteristic of the traditional solution phasecoupling reactions. (Church, et al. Biochemistry 1990, 29, 6827. He, etal. J. Am. Chem. Soc. 1993, 115, 7061.)

The process of expanding the 2:1 polyamide-DNA motif to include longersequences recognized by increasingly complex polyamides is demanding.For example, using previously described multi-step solution phasechemistry, the total synthesis of the hairpin polyamidesImPyPy-γ-PyPyPy-Dp required more than a month's effort.

The chemical structures of the polyamide of the prior artImPyPy-γ-PyPyPy-Dp, and the optimized hairpin polyamideImPyPy-γ-PyPyPy-β-Dp provided by the instant invention are shown below:

Hereinafter hairpins may be shown as chemical structures binding to aschematic representation of the minor groove. An abbreviatedrepresentation may alternatively be used wherein, imidazole rings arerepresented as filled circles, pyrrole rings are represented as unfilledcircles, β-alanine is represented as a diamond, Glycine is representedas a triangle, amide bonds are represented as lines, γ-aminobutyric acidis represented as a curved line, and the positively chargeddimthylaminopropylamide is represented with a (+). An example of bothnotations is shown below for the optimized 6-ring hairpin polyamideImPyPy-γ-PyPyPy-β-Dp binding to a cognate 5′-TGTTA-3′ site:

It has been shown that the Py/Py pair is approximately degenerate forrecognition of A,T base pairs, affording generality with regard totargeting sequences of mixed A•T/T•A composition. (White, et al.Biochemistry 35, 12532-12537 (1996)). To test the extent of thisdegeneracy, the affinity of the hairpin polyamide ImPyPy-γ-PyPyPy-β-Dpwas measured for eight possible five base pair 5′-TG(A,T)₃-3′ matchsites. Quantitative DNase I footprint titration experiments reveal thatImPyPy-γ-PyPyPy-β-Dp binds all eight 5′-TG(A,T)₃-3′ target sites withonly a 12-fold difference in the equilibrium association constantsbetween the strongest site, 5′-TGTTT-3′ (K_(a)=2.1×10⁸ M⁻¹) and theweakest site, 5′-TGAAT-3′ (K_(a)=1.8×10⁷ M⁻¹) (10 mM Tris•HCl, 10 mMKCl, 10 mM MgCl₂, 5 mM CaCl₂, pH 7.0, 22° C.).

Sites are recognized with decreasing affinity:5′-TGTTT-3′>5′-TGTTA-3′>5′-TGTAA-3′>5′-TGTAT-3′>5′-TGATT-3′>5′-TGATA-3′>5′-TGAAA-3′>5′-TGAAT-3′as shown in schematic form below:

These results indicate that all sites of the form 5′-TG(A,T)₃-3′ arestructurally compatible with polyamide-DNA complex formation. However,the affinities of ImPyPy-γ-PyPyPy-β-Dp for 5′-TG(A,T)₃-3′ binding sitesmay be grouped into two sets according to sequence composition:5′-TGT(A,T)₂-3′ and 5′-TGA(A,T)₂-3′. ImPyPy-γ-PyPyPy-β-Dp binds5′-TGT(A,T)₂-3′ sites with between 2-fold and 12-fold higher affinitythan 5′-TGA(A,T)₂-3′ sites. Therefore binding sites containing 5′-GT-3′steps may be preferred over those containing 5′-GA-3′ steps fortherapeutic targets.

These results indicate that at least a 10-fold range of bindingaffinities and sequence specificities will be observed for a polyamidebinding to a designated set of match sites containing A•T base pairs.This relatively small range indicates that, in contrast to the Im/Pypair which may distinguish G•C from C•G and both of these from A•T/T•Abase pairs, the Py/Py pair appears not to distinguish A•T from T•A basepairs. The similarity of the polyamide binding affinities for the eight5′-TG(A,T)₃-3′ match sites reflects a limit to the specificity of thehairpin polyamide binding motif. Because G•C is distinct from C•G, themost specific recognition will be observed for G•C rich sequences.

In principle, individual polyamide subunits can recognize DNA with twopossible binding orientations. Recognition of 5′-TGTTA-3′ by a polyamideof core sequence composition ImPyPy-γ-PyPyPy places the N-terminus ofeach polyamide subunit at the 5′-side of each recognized DNA strand.Placement of the polyamide N-terminus at the 3′ side of each recognizedstrand would result in targeting of a 5′-TCTTA-3′ sequence. Each bindingorientation represents a unique and distinguishable hairpin fold.Subunit orientation preference was not defined by the prior art,however, in order to successfully apply the pairing rules towardspolyamide design, a single predictable subunit binding orientation mustbe preferred.

A schematic model of two possible hairpin polyamide DNA-bindingorientations is shown below:

It has been observed that a 30-fold (2 kcal/mol) binding-orientationpreference exists for a 6-ring hairpin polyamide binding with theN-terminal end of each subunit located towards the 5′-side of therespective targeted DNA strand. The pyrrole-imidazole polyamideDNA-binding orientation preference defines a second order design rulewhich must be considered for successful application of the pairing rulesfor polyamide design.

The potential degeneracy of the Im/Py and Py/Im pairs for recognition ofGC and CG has not been sufficiently addressed by the prior art. Theexocyclic amine group of guanine is symmetrically placed in the floor ofthe minor groove, and will therefore be displayed in the same locationfor CG and GC base pairs. Single mismatch binding sites described inprior art were exclusively GC to AT substitutions. It was unclear tothose skilled in the art whether sequences which differ by a single GCto CG substitution would be discriminated by the pyrrole-imidazolepolyamide-DNA motif. The rapid design of new polyamides for elucidationcomplete pairing rules was aided by the discovery that the hairpin-polyamide motif is compatible with solid phase synthetic methods.

A series of four polyamides were prepared: ImPyPy-γ-PyPyPy-β-Dp,ImImPy-γ-PyPyPy-β-Dp, ImPyPy-γ-PyImPy-β-Dp, and ImImPy-γ-PyImPy-β-Dp.Each polyamide places a Py/Py, Im/Py, Py/Im, or Im/Im pair oppositeeither a T/A or G/C base pair in eight possible ring pairing-base paircombinations. The structure of four hairpin polyamides, which differ inthe central ring pairings, are shown in FIG. 2.

It was determined that Im/Py and Py/Im pairs effectively discriminateGC from CG base pairs, respectively and that a Im/Im pairingrepresents an energetically unfavored pairing. Quantitative DNaseIfootprinting experiments reveal energetics of the four possiblepyrrole-imidazole polyamide ring pairings. Py/Py is found to bindpreferably to AT/TA>>GC/CG, Im/Py binds GC>>TA/AT >CG, and Im/Imdoes not bind GC/CG or AT/TA. A schematic representation of theeight possible ring pairing-base pair interactions is shown below:

These results shown that GC and CG base pairs may be distinguished inthe minor groove, while the energetic penalty for formation of an Im/Impairing provides a basis for design of specific unlinked overlappedpolyamide complexes as will become evident below.

It has been determined that the 6-ring hairpin polyamide motif providesa versatile template for recognition of a wide variety of sequences inthe DNA minor groove. (Parks, et al. J. Am. Chem. Soc., 118, 6153(1996); Szewczyk, et al. Angew. Chemie, 35, 1487-1489 (1996); Swalley,et al. J. Am. Chem. Soc. 118, 8198-8206(1996)). Six-ring hairpinpolyamides recognize their cognate sites with affinities ranging from1×10⁷ M⁻¹ to 1×10⁸ M⁻¹ and specificity against single base pair mismatchsites ranging from 2-fold to 60-fold.

A schematic of nine 6-ring hairpin polyamides recognizing cognate 5 basepair sites is shown below:

The broad sequence repertoire recognized by the 6-ring hairpin motifrepresents a significant advance in ligand design. However, no 6-ringhairpin polyamide has been identified which recognizes a target sitewith subnanomolar affinity.

To determine the effect of polyamide length on binding site size,binding affinity, and sequence specificity, a series of six polyamidescontaining three to eight rings was synthesized. (Kelly, et al. Proc.Natl. Acad. Sci. U.S.A. 93, 6981-6985 (1996).) The series is based onImPyPy-Dp with pyrrolecarboxamide moieties added sequentially to theC-termini to afford ImPyPyPy-Dp, ImPyPyPyPy-Dp, ImPyPyPyPyPy-Dp,ImPyPyPyPyPyPy-Dp, and ImPyPyPyPyPyPyPy-Dp which are designed to bind 5to 10 base pair sites, respectively as side-by-side antiparellel dimers.DNA binding sites are based on a 5′-TGACA-3′ core sequence and containsequential A,T inserts in the center of the binding site that will berecognized by the additional pyrrole carboxamides. Chemical structuresof the polyamides are shown in FIG. 3.

It was determined that polyamides based on 4 or 5-ring subunits areoptimal, and that subunits must not contain more than 5 consecutiverings. Binding affinity reaches a maximum value for the five ringpolyamide ImPyPyPyPy-Dp and addition of up to two additionalpyrrolecarboxamides has no effect on the observed association constant(Table 2). Furthermore, sequence specificity decreases as the length ofthe polyamides increases beyond five rings.

TABLE 2 Table 1* polyamide-DNA complex association constantspecificity^(‡)

1.3 × 10⁵ M⁻¹ 6.5-fold

8.5 × 10⁶ M⁻¹ 5.3-fold

4.5 × 10⁷ M⁻¹ 5.7-fold

5.3 × 10⁷ M⁻¹ 2.7-fold

4.7 × 10⁷ M⁻¹ 2.8-fold

 <2 × 10⁷ M⁻¹ 1-fold *Values reported are the mean values from at leastthree footprint titration experiments. The assays were performed at 22°C., pH 7.0, in the presence of 10 mM TrisHCl, 10 mM KCl, 10 mM MgCl₂ and5 mM CaCl₂. ^(‡)Defined as the ratio of the match site affinity to theaffinity of the single base pair mismatch site.

These results, specifically the failure of an eight-ring polyamide torecognize a 10-base pair target site suggested that a new class ofpolyamides was needed was needed for extension of the 2:1 polyamide-DNAmotif to sequences longer than 9 base pairs. The present inventionprovides for the replacement of a central pyrrole or imidazole aminoacid with a more flexible amino acid subunit, thus allowing theantiparallel dimer to reset the register for continued gain in affinityand specificity.

To identify a flexible linker amino acid, four polyamides of the formulaImPyPy-X-PyPyPy-Dp where X=Py, G (glycine), β, or γ, respectively, weresynthesized and their equilibrium association constants determined for5′-TGTTAAACA-3′ (9 base pair) sites. (Trauger, et al. J. Am. Chem. Soc.,118, 6160 (1996).)

The structure of polyamides based on ImPyPy and PyPyPy-Dp subunitslinked by pyrrole or flexible glycine or β-alanine linkers are shownbelow:

It was determined that β-alanine is an optimal linker for joiningpolyamide subunits in an extended conformation, providing a usefulstructural motif for the design of new polyamides targeted to sequenceslonger than 7 base pairs. The β-alanine-linked compoundImPyPy-β-PyPyPy-Dp has the highest binding affinity of the fourpolyamides, binding the 9 bp site 5′-TGTTAAACA-3′(K_(a)=8×10⁸ M⁻¹) withaffinities higher than the formally N-methylpyrrole-linked polyamideImPyPy-Py-PyPyPy-Dp by a factor of −10.

Solid phase synthesis involves the stepwise assembly of a molecule whileone end is covalently anchored to an insoluble matrix at all stages ofthe synthesis. (Merrifield, J. Am. Chem. Soc. 85, 2149-2154 (1963);Merrifield, Science 232, 341-347.) The solid phase approach has beensuccessfully developed for a variety of proteins' (Gutte, et al. 246,1922-1941 (1971)), oligonucleotides (Ként, S. B. H. Ann. Rev. Biochem.57, 957-989 (1988); Caruthers, et al. Methods In Enzymology 154, 287-313(1987); Caruthers, M. H. Acc. Chem. Res. 24, 278-284 (1991)) peptoids,(Simon, et al. Proc. Natl Acad. Sci. U.S.A., 89, 9367-9371 (1992);Zuckermann, et al. J. Am. Chem. Soc. 114 10646-10647 (1992)),oligosacharides (Science 269, 202-204 (1995); Science 260, 1307-1309(1993)), and small non-polymeric molecules (Ellman, J. A. Acc. Chem.Res. 29, 132-143.) General protocols have been developed for manual andmachine-assisted Boc-chemistry solid phase synthesis of pyrrole-imidazole polyamides. (Baird and Dervan, J. Am. Chem. Soc., 118, 6141(1996)). More specifically, the following components were developed: (1)a synthesis which provides large quantities of appropriately protectedmonomer or dimer building blocks in high purity, (2) optimized protocolsfor forming an amide in high yield from a support bound aromatic amineand an aromatic carboxylic acid, (3) methods for monitoring reactions onthe solid support, (4) a stable resin linkage agent that can be cleavedin high yield upon completion of the synthesis. Solid phase synthesisprotocols for pyrrole- imidazole polyamides reduce the syntheticinvestment from months to days.

A representative solid phase synthesis of a polyamide is shown in FIG.4. Polyamides containing more than 4 residues are preferably prepared bysolid phase methodology. For solid phase synthesis, the polyamide isattached to an insoluble matrix by a linkage which is cleaved by asingle step process which introduces a positive charge into thepolyamide. The addition of an aliphatic amino acid at the C-terminus ofthe pyrrole- imidazole polyamides allows the use ofBoc-β-alanine-Pam-Resin resin which is commercially available inappropriate substitution levels (0.2 mmol/gram) (Mitchell, et al. J.Org. Chem. 1978, 43, 2845.) Aminolysis of the resin ester linkageprovides a simple and efficient method for cleaving the polyamide fromthe support.

Solid phase polyamide synthesis protocols were modified from the in situneutralization Boc-chemistry protocols recently reported by Kent andcoworkers. (Schnolzer, et al. Int. J. Peptide. Protein. Res. 1992, 40,180: Milton, et al. Science 1992, 256,1445.) Coupling cycles are rapid,72 min per residue for manual synthesis or 180 min per residue formachine-assisted synthesis, and require no special precautions beyondthose used for ordinary solid phase peptide synthesis. The manual solidphase protocol for synthesis of pyrrole-imidazole polyamides has beenadapted for use on a ABI 430A peptide synthesizer. Stepwise cleavage ofa sample of resin and analysis by HPLC indicates that high stepwiseyields (>99%) are routinely achieved.

The large number of polyamides made available by solid phase syntheticmethodology makes possible the elucidation of the rules necessary fordevelopment of polyamides which bind DNA with subnanomolar affinities.Cleavage of the polyamide from the resin with a primary diamine providesa polyamide having an unmodified primary amine group. The amine groupmay then be modified with an activated carboxylic acid or bynucleophilic aromatic substitution to provide a bifunctional polyamide.

Standard techniques available to one skilled in the art may be used todetermine the DNA binding properties of novel pyrrole-imidazolepolyamides. Affinity cleaving titration experiments ((25 mMTris-Acetate, 20 mM NaCl, 100 mM bp calf thymus DNA, pH 7, 22° C., 10 mMDTT, 10 mM Fe(II)) using polyamides modified with EDTAFe(II) at theC-terminus are used to determine oriented binding. MPEFe(II)footprinting experiments (Hertzberg and Dervan, J. Am. Chem. Soc., 104,313 (1982); Van Dyke and Dervan, Biochemistry, 22, 2373 (1983); Van Dykeand Dervan, Nucleic Acids Res., 11, 5555 (1983); Hertzberg and Dervan,Biochemistry, 23, 3934 (1984)) (25 mM Tris-acetate, 10 mM NaCl, 100 μMcalf thymus DNA, 5 mM DTT, pH 7.0 and 22° C.) are used to determinebinding site size. Quantitative DNaseI footprinting (Brenowitz, et al.(1986). Methods Enzymol. 130, 132-181.; Fox and Waring (1984). NucleicAcids Res. 12, 9271-9285 Brenowitz, M., Senear, D. F., Shea, M. A. &Ackers, G. K. (1986); Proc. Natl. Acad. Sci. U.S.A. 83, 8462-8466.) (10mM Tris-HCl, 10 mM KCl, 10 mM MgCl₂, and 5 mM CaCl₂, pH 7.0, 22° C.)reveals the equilibrium association constants for binding to match andmismatch sites. All footprinting experiments are performed on 3′ and5′³²P end restriction fragments derived from plasmids. 3′-shiftedcleavage patterns are consistent with location of the polyamide in theminor groove.

Tert-butoxycarbonylaminoacyl-4-(oxymethyl)phenyl-acetamidomethyl-resin(PAM resin) is commercially available and cleaved in high yield byaminolysis with primary amines. (Mitchell, A. R.; Kent, S. B. H.,Engelhard, M.; Merifield, R. B., J. Org. Chem. 43, 2845.) Insertion of aC-terminal aliphatic amino acid residue makes the hairpin- polyamidemotif compatible with solid phase synthetic methods, allowing the rapiddesign of new polyamides. This result sets the stage for the elucidationof the limits of hairpin motif with regards to binding site size,binding affinity, and sequence specificity.

A schematic representation of the recognition of a nine base pair targetsite, by a polyamide containing a β-spring is shown below:

The binding data for ImPyPy-γ-PyPyPy-Dp, which was shown previously tobind DNA in a “hairpin” conformation, indicates that γ-aminobutyric aciddoes not effectively link polyamide subunits in an extendedconformation. The discovery of β-alanine as an effective linker forjoining polyamide subunits in an extended conformation, provides auseful structural motif for the design of new polyamides based onsubunits <5-rings targeted to sites longer than 7 bp.

At least two distinct binding modes are expected to form for theImPyPy-X-PyPyPy-Dp polyamides described above that bind in an extendedconformation. These binding modes as “slipped” and “overlapped”. In theoverlapped (9 base pair) binding mode, two ImPyPy-X-PyPyPy-Dp polyamidesbind directly opposite one another. The “slipped” (13 base pair) bindingmode integrates the 2:1 and 1:1 polyamide-DNA binding motifs at a singlesite. In this binding mode, the ImPyPy moieties of twoImPyPy-X-PyPyPy-Dp polyamides bind the central 5′-AGACA-3′ sequence in a2:1 manner as in the ImPyPy homodimer, and the PyPyPy moieties of thepolyamides bind to A,T flanking sequences as in the 1:1 complexes ofdistamycin.

A schematic model of the “slipped” and “overlapped” binding modes isshown below.

The present invention provides β-alanine as an optimal linker forjoining polyamide subunits in a “slipped” extended conformation,providing a structural motif whereby a MW≈900 polyamide recognizes a 13base-pair DNA sequence. The β-alanine-linked compound ImPyPy-β-PyPyPy-Dpbinds to a 13 bp 5′-AAAAAGACAAAAA-3′ site with an association constant.K_(a)=5×10⁹ M⁻¹, that is higher than the formally N-methylpyrrole-linkedpolyamide ImPyPy-Py-PyPyPy-Dp by a factor of ˜85.

As described above, γ-aminobutyric acid, and preferably β-alanine,effectively link polyamides in hairpin and extended conformations,respectively. It has also been demonstrated that γ-aminobutyric does notoptimally link polyamide subunits in extended conformations, and thatβ-alanine does not optimally link polyamide subunits in hairpinconformations. These results suggested that γ-aminobutyric acid andβ-alanine could be combined within a single polyamide with predictableresults. (Trauger, et al, Chem. & Biol., 3, 369 (1996)).

It has been determined that the nine-ring “extended hairpin” polyamideImPyPy-γ-ImPyPy-β-PyPyPy-G-Dp binds a 9-bp target site 5′-AAAAAGACA-3′at 0.05 nM concentration, an increase in affinity relative to thesix-ring hairpin polyamide ImPyPy-γ-ImPyPy-β-Dp of ˜400-fold. Theseresults provide a strategy for increasing the DNA-binding affinity ofhairpin polyamides into the subnanomolar range. Furthermore, as willbecome evident below, many important DNA binding transcription factorssuch as TBP and homeodomain proteins have A,T rich consensus sequences.Extended hairpin polyamides provide a general method by which apolyamide may interfere with protein-DNA interactions by recognizing aunique sequence adjacent to certain protein binding sites. A schematicbinding model of extended hairpin polyamide recognition of a 9 base pairsequence is shown below:

Provided herein are extended hairpin polyamide motifs that provideversatile templates for recognition of a wide variety of sequences inthe DNA minor groove. Extended hairpin polyamides recognize their 9 to13 base pair sites target site with affinities ranging from 1×10⁸ M⁻¹ to>5×10¹⁰ M⁻¹ and specificity against single base pair mismatch sitesranging from 5-fold to 60-fold. A schematic of nine extended hairpinpolyamides containing 9 to 12 rings and recognizing 9 to 13 base pairtarget sites is shown in FIG. 5.

Provided herein is an endonuclease protection assay to measure the rateof polyamide-DNA complex formation. Such an assay may comprise a labeledrestriction fragment comprising a polyamide binding site that overlaps arestriction endonuclease cleavage site. Cleavage by the cognate isprevented when the overlapping polyamide binding site is occupied by thepolyamide. As a control, a second labeled DNA fragment may be thatcontains the restriction site, but lacks the overlapping polyamidebinding site. The rate of polyamide association with its target bindingsite may be assessed by incubating the solutions of the polyamide withthe labeled target and reference fragments for a sufficient timerperiod. Using the experimental conditions provided herein, the referencesite is nearly completely digested, but protection at the target site isobserved and can be correlated with polyamide concentration and the timeof equilibration. Similarly, the dissociation rate is analyzed by addingan excess of unlabeled competitor DNA to an equilibrated solution of thelabeled DNA fragments and polyamide. Addition of the competitor reducesthe concentration of free polyamide to zero. The rate at with polyamidedissociation occurs from the target site on the labeled fragment can befollowed by the rate of loss of protection from restriction enzymedigestion as the re-equilibration time is increased.

First generation six-ring hairpin polyamides bind DNA with associationconstants of approximately 1×10⁸ M⁻¹ (FIG. 6) The observation thatunlinked four-ring polyamides form 2:1 complexes with 70-fold-higheraffinity relative to three-ring polyamides suggested an eight-ringhairpin polyamide motif for recognition of DNA at subnanomolarconcentration. The present inventor has shown that two eight-ringpyrrole-imidazole polyamides differing in sequence by a single aminoacid bind specifically to respective six base pair target sites whichdiffer in sequence by a single base pair. (Trauger, et al. Nature, 382,559-561 (1996)). Binding is observed at subnanomolar concentrations ofligand.

DNA-binding affinities were determined for two eight-ring hairpinpolyamides. ImPyPyPy-γ-ImPyPyPy-β-Dp and ImPyPyPy-γ-PyPyPyPy-β-Dp, whichdiffer by a single amino acid, for two 6 base pair (bp) target sites,5′-AGTACT-3′ and 5′-AGTATT-3′, which differ by a single base pair. Basedon the pairing rules for polyamide-DNA complexes, the sites 5′-AGTACA-3′and 5′-AGTATT-3′ are for ImPyPyPy-γ-ImPyPyPy-β-Dp “match” and “singlebase pair mismatch” sites, respectively, and for polyamideImPyPyPy-γ-PyPyPyPy-β-Dp “single base pair mismatch” and “match” sites,respectively. Binding models for 5′-AGTACT-3′ and 5′-AGTATT-3′ incomplex with ImPyPyPy-γ-ImPyPyPy-β-Dp and ImPyPyPy-γ-PyPyPyPy-β-Dp areshown in FIG. 7.

ImPyPyPy-γ-ImPyPyPy-β-Dp and ImPyPyPy-γ-PyPyPyPy-0β-Dp were synthesizedby solid phase methods and purified by reversed phase HPLC. Equilibriumassociation constants for match and mismatch six base pair binding siteson a 3′-³²P-labeled 229 bp restriction fragment were determined byquantitative DNase I footprint titration experiments.ImPyPyPy-γ-ImPyPyPy-β-Dp binds its match site 5′-AGTACT-3′ at 0.03 nMconcentration and its single base pair mismatch site 5′-AGTATT-3′ withnearly 100-fold lower affinity. ImPyPyPy-γ-PyPyPyPy-β-Dp binds itsdesignated match site 5′-AGTATT-3′ at 0.3 nM concentration and itssingle base pair mismatch site 5′-AGTACT-3′ with nearly 10-fold loweraffinity. The specificity of ImPyPyPy-γ-ImPyPyPy-β-Dp andImPyPyPy-γ-PyPyPyPy-β-Dp for their respective match sites results fromvery small structural changes. Replacing a single nitrogen atom inImPyPyPy-γ-ImPyPyPy-β-Dp with C—H reduces the affinity of thepolyamide5′-AGTACT-3′ complex by −75-fold representing a free energydifference of −2.5 kcal/mole. Similarly, replacing a C—H inImPyPyPy-γ-PyPyPyPy-β-Dp with N reduces the affinity of thepolyamide5′-AGTATT-3′ complex −10-fold, a loss in binding energy of−1.3 kcal/mol.

These results show that using a simple molecular shape and a two letteraromatic amino acid code, pyrrole-imidazole polyamides can achieveaffinities and specificities comparable to DNA-binding proteins. Itremained to be determined if additional motifs could be discovered toprovide polyamides with subnanomolar binding affinities.

It has been suggested that pyrrole-imidazole polyamides would bind G/Crich sequences with low binding affinity due to steric hindrance withthe exocyclic amines of the guanine bases. It has also been noted thatthe lower negative electrostatic potential of a G/C rich minor grooverelative to an A, T rich minor groove might prohibit high affinitybinding. (Pullman, et al. Quarterly Reviews of Biophysics. (1981) 14,289-380; Pullman, B. Advances in Drug Research. (1989) 18, 1-113.Manning, G. S. Q. Rev. of Biophysics. (1978) 11, 179-246; Honig andNicholls. Science (1995) 268, 1144.) It has been found that an 8-ringhairpin polyamide can recognize a G/C rich target sequence withsubnanomolar affinity.

Schematic binding models of eight-ring hairpin polyamides designed forrecognition of 5′-(A/T) (G/C)₄ (A/T)-3′ sequences.

To examine whether a core sequence of purely G,C base pairs could berecognized with high affinity and specificity, three eight-ring hairpinpolyamides which differ only by the arrangement of pyrrole and imidazoleamino acids. ImImPyPy-γ-ImImPyPy-β-Dp, ImPyImPy-γ-ImPyImPy-β-Dp, andImImImIm-γ-PyPyPyPy-β-Dp were designed for recognition of three coresequences consisting of solely G,C base pairs. DNase I footprinttitrations allow the determination of equilibrium association constants(K_(a)) for each polyamide. ImImPyPy-γ-ImImPyPy-β-Dp binds the matchsite 5⁻-TGGCCA-3′ with an equilibrium association constants ofK_(a)=1×10¹⁰ M⁻¹ (10 mM TrisHCl, 10 mM KCl, 10 mM MgCl₂ and 5 mM CaCl₂,pH 7.0 and 22° C.). The two designed double base pair mismatchsequences, 5⁻-TGCGCA-3′ and 5′-TGGGGA-3′, are bound with at least200-fold reduced affinity. ImPyImPy-γ-ImPyImPy-β-Dp binds the site5′-TGCGCA-3′ with a K_(a)=4×10⁷ M⁻¹ with 4-fold specificity, andImImImIm-γ-PyPyPyPy-β-Dp binds the site 5′-TGGGGA-3′ with a K_(a)=3×10⁷M⁻¹ with 6-fold specificity.

These results indicate that the positioning of the Im amino acids have aprofound effect on the binding affinities of pyrrole-imidazolepolyamides. More specifically these results indicate that bindingaffinity could be restored by the design of hairpin polyamides where apyrrole ring has been substituted by more flexible spacer amino acidsuch as β-alanine.

It has been found that replacement of a pyrrole residue with a β-alaninespacer residue in each subunit of ImPyImPy-γ-ImPyImPy-β-Dp provides aneight residue hairpin polyamide, Im-β-ImPy-γ-Im-β-ImPy-β-Dp, whichrecognizes 5′-TGCGCA-3′ sequences with subnanomolar affinities.

Structures and schematic binding models for the eight ring hairpinpolyamide ImPyImPy-γ-ImPyImPy-β-Dp and the eight residue hairpinpolyamide Im-β-ImPy-γ-Im-0β-ImPy-β-Dp are shown in FIG. 8.

It has been found that the four ring hairpin polyamide motif provides aversatile template for recognition of a wide variety of sequences in theDNA minor groove. Eight ring and residue hairpin polyamides recognize 6base pair target sites with affinities ranging from 1×10⁷ M⁻¹ to >1×10¹⁰M⁻¹ and specificity against single base pair mismatch sites ranging from2-fold to >100-fold. A schematic of fifteen 8-residue hairpin polyamidesrecognizing 6 base pair target sites is shown in FIG. 9.

First generation fully overlapped β-linked polyamides based on threering subunits bind DNA with association constants of approximately 8×10⁸M⁻¹. The observation that unlinked four-ring polyamides form 2:1complexes with 70-fold-higher affinity relative to three-ring polyamidessuggested a fully overlapped 8-ring 4-β-4 polyamide motif forrecognition of 11 base pairs of DNA at subnanomolar concentration. Thechemical structures of three 4β-4 polyamides are shown in FIG. 10.

It has been found that three eight ring 4β-4 pyrrole-imidazolepolyamide, ImImImPy-β-PyPyPyPy-β-Dp, ImImPyPy-β-PyPyPyPy-β-Dp andImPyPyPy-β-PyPyPyPy-β-Dp specifically recognize targeted5′-AGGGATTCCCT-3′, 5′-AGGTATTATCCT-3′ and 5′-AGTAATTTACT-3′ sites,respectively. DNase I footprint titrations reveal that each polyamidebinds its respective target site at subnanomolar conentrations withequilibrium association constants ranging from K_(a)=7×10⁹ M⁻¹ to K_(a)5×10¹⁰ M⁻¹, and with 7 to 30-fold specificity over double base pairmismatch sites.

The ability of 3β-3 and 4β-4 polyamides to recognize both “slipped” and“overlapped” complexes for recognition of two separate classes of targetsites represents a limit to the sequence specificity of the β-extendedpolyamide motif. The discovery that a Im/Im polyamide pairing isdisfavored, suggests that the 4β-4 polyamide ImImImPy-β-PyPyPyPy-β-Dpshould bind preferentially in the fully overlapped polyamide motif. Aschematic representation of the recognition of three targeted DNA sitesby three 4β-4 polyamides is shown in FIG. 11.

The 4γ-4 polyamide ImImImPy-γ-PyPyPyPy-β-Dp binds a 5′-AGGGAA-3′ targetsite in a hairpin conformation with an association constant ofK_(a)=4×10⁸. The 4γ-4 polyamide ImImImPy-γ-PyPyPyPy-β-Dp is related tothe 4-β-4 polyamide ImImImPy-β-PyPyPyPy-β-Dp by deletion of a singlemethylene unit (MW=14) from the linker region. The γ and β linkersspecificity turn and extended binding respectively and enlarge targetedbinding site size from 6 to 11 base pairs, resulting in a 2.1 kcal/molenhancement in binding energy. These results, the specific recognitionof a G,C-rich 11 base pair sequence, represent a significant advance inthe development of general DNA-binding that can recognize a single sitein the human genome.

It has been determined that there exists at least a 20-fold preferencefor placement of a β/β pair opposite an AT or TA base pair relative toa GC or CG base pair. Quantitative DNase I footprint titrationexperiments reveal that ImImImPy-β-PyPyPyPy-β-Dp binds the designedmatch site 5′-AGGGAATCCCT-3′ with an equilibrium association constant ofK_(a) 1.4×10¹⁰ M⁻¹ and the single base pair β/β mismatch sequence5′-AGGGAGTCCCT-3′ with at least 20-fold lower affinity (K_(a)=6.9×10⁸M⁻¹). These results implicate the β/β combination as both a flexiblespacer unit and a sequence-specific DNA binding element. The specificityof the β/β pairing reveals an additional pairing rule pivotal to thedesign of polyamides for recognition of longer binding sites. Aschematic model of placement of the β/β pair opposite, G,C or A,T basepairs is shown in FIG. 12.

It has been found that the extended, fully overlapped polyamide-DNAmotif, provides a versatile template for recognition of symmetricsequences containing from 9 to 13 base pairs in the minor groove.Equilibrium association constants for cooperative complex formationrange from K_(a)=1×10⁷ M⁻¹ to K_(a)>1×10¹¹ M⁻¹. Specificities have beenfound to range from 2-fold to >20-fold for discrimination of single basepair mismatch sites. A schematic representation of several β-linkedfully overlapped polyamide complexes is shown in FIG. 13.

To further expand the targetable binding site size and sequencerepertoire available to the hairpin polyamide motif, two polyamidescontaining either two or three Im amino acid residues,ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp and ImImPyPyPy-γ-ImPyPyPyPy-β-Dp, wereprepared by solid phase synthetic methodology and their DNA bindingproperties analyzed. The structures of two 10-ring hairpin polyamidesare shown in FIG. 14.

It has been shown that that ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp binds theformal 7 bp match sequence 5′-TGTAACA-3′ with an equilibrium associationconstant (K_(a)) of K_(a) 1.2×10¹⁰ M⁻¹ and the single base pair mismatchsequence 5′-TGGACA-3′ with K_(a)=6.8×10⁸ M⁻¹. (10 mM Tris.Hcl, 10 mMKCl, 10 mM MgCl₂, 5 mM CaCl₂, pH 7.0, 22° C.).ImImPyPyPy-γ-ImPyPyPyPy-β-Dp, which differs fromImPyPyPyPy-γ-ImPyPyPyPy-β-Dp by a single amino acid substitution bindsits formal match sequence 5′-TGGAACA-3′ with an equilibrium associationconstant of K_(a)=3.6×10⁹ M⁻¹ and its corresponding single base pairmismatch sequence 5′-TGTAACA-3′ with K_(a)<1×10⁷ M⁻¹. The replacement ofa single electron lone-pair with a hydrogen atom within a −1500 MWpolyamide is found to modulate affinity and specificity by more than anorder of magnitude. Sequence-specific recognition of a 7 bp target siteby a ten-ring hairpin polyamide at subnanomolar concentration expandsthe effective targetable sequence repertoire of the pyrrole-imidazolepolyamide-DNA motif.

A schematic model of two 10-ring hairpin polyamides recognizing matchand mismatch 7 base pair sequences is shown below:

The specificity of that ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp and thatImImPyPyPy-γ-ImPyPyPyPy-β-Dp for their respective match sites resultsfrom very small structural changes. Replacing a single C—H in thatImPyPyPyPy-γ-ImPyPyPyPy-β-Dp, with a nitrogen atom as in thatImImPyPyPy-γ-ImPyPyPyPy-β-Dp reduces the affinity of theImImPyPyPy-γ-ImPyPyPyPy-β-Dp.5′-TGTAACA-3′ complex relative to theImPyPyPyPy-γ-ImPyPyPyPy-β-Dp.5′-TGTAACA-3′ complex by >300-fold, a freeenergy difference of at least 4 kcal/mol. Similarly, replacing a N inthat ImImPyPyPy-γ-ImPyPyPyPy-β-Dp with a C—H as in thatImPyPyPyPy-γ-ImPyPyPyPy-β-Dp, reduces the affinity of theImPyPyPyPy-γ-ImPyPyPyPy-β-Dp.5′-TGGAACA-3′ complex relative to theImImPyPyPy-γ-ImPyPyPyPy-β-Dp.5′-TGGAACA-3′ complex by a factor of5-fold, a loss in binding energy of −1 kcal/mol. The reduced overallspecificity and binding affinity of that ImImPyPyPy-γ-ImPyPyPy-β-Dprelative to that ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp most likely results fromthe presence of a 5′-GA-3′ step in the designated target site.

A polyamide, ImPy-β-ImPy-γ-ImPy-β-ImPy-β-Dp, based on β-alanine linked2-ring subunits was prepared to target a seven basepair region adjacentto a binding site for the transcription factor TBP in a conserved HIVgene-promoter sequence. The polyamide was designed based on the pairingrules described herein, and was found to recognize its designated5′-TGCTGCA-3′ target sequence with a binding affinity of K_(a)=3.6×10⁹M⁻¹. An isomeric mismatched polyamide, ImPy-β-ImPy-γ-ImPy-β-ImPy-β-Dp,which differs only by the position of the Py and Im amino acids withinthe 2-β-2-γ-2-β-2 molecular template binds the targeted 5-TGCTGCA-3′sequence with 100-fold reduced affinity. A schematic representation of apolyamide and a control polyamide which are molecular isomers, yetdiscriminate a 7-base pair sequence of an HIV gene promoter with a100-fold specificity is shown in FIG. 15.

These results reveal that hairpin polyamides based on 5-ring subunitsprovide a useful structural motif for the recognition of 7 bp bindingsites at sunanomolar concentrations. For targeting 5′-WGWWWCW-3′sequence a 5-γ-5 polyamide, ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp based on 2γ-aminobutyric acid linked 5-ring subunits is preferred over thecorresponding β-substituted, 2-β-2-γ-2-β-2 polyamideImPy-β-PyPy-γ-ImPy-β-PyPy-β-Dp. For targeting 5′-WGCWGCW-3′ and5′-WGGWGGW-3′ sequences the respective β-substituted, 2-β-2-γ-2-β-2polyamides ImPy-β-ImPy-γ-ImPy-β-ImPy-β-Dp andImIm-β-ImIm-γ-PyPy-β-PyPy-β-Dp are preferred over the respective 5-β-5polyamides, ImPyPyImPy-γ-ImPyPyImPy-β-Dp andImImPyImIm-γ-PyPyPyPyPy-β-Dp based on γ-aminobutyric acid linked 5-ringsubunits. A series of hairpin polyamides which recognize 7 base pairtarget sites are shown in FIG. 16.

The present inventor has discovered that a β/β pairing is preferred to aPy/β pairing for extension of the targetable binding site size of thehairpin polyamide motif. Three “12-ring hairpin” polyamides,ImPyPyPyPyPy-γ-ImPyPyPyPyPy-β-Dp, ImPyPy-β-PyPy-γ-ImPyPy-β-PyPy-β-Dp andImPy-β-PyPyPy-γ-ImPyPy-β-PyPy-β-Dp were synthesized by solid phasesynthetic methodology.

TABLE 3 Mismatch Mismatch Specificity

6-fold

6-fold

55-fold

DNase I footprint titrations reveal that the hairpin polyamide based on6-ring subunits, ImPyPyPyPyPy-γ-ImPyPyPyPyPy-β-Dp, binds the formal 8 bpmatch sequence 5′-TGTTAACA-3′ with an equilibrium association constant(K_(a)) of K_(a)=4×10⁹ M⁻¹ and the single base pair mismatch sequence5′-TGTGAACA-3′ with K_(a)=2×10⁸ M⁻¹. ImPyPy-β-PyPy-γ-ImPyPy-β-PyPy-β-Dpwhich differs from ImPyPyPyPyPy-γ-ImPyPyPyPyPy-β-Dp by substitution oftwo flexible aliphatic amino acid residues for two pyrrole rings, bindsa 5′-TGTTAACA-3′ match site K_(a)=2×10¹⁰ M⁻¹ and a 5′-TGTGAACA-3′mismatch with K_(a)=1×10⁹ M⁻¹. ImPy-β-PyPyPy-γ-ImPyPy-β-PyPy-β-Dp bindsa 5′-TGTTAACA-3′ match site with an equilibrium association constant ofK_(a) 1×10¹¹ and a single base pair mismatch sequence 5′-TGTGAACA-3′with K_(a)<1×10⁹. (Table II). These results expand the targetablebinding site size accessible to the hairpin polyamide motif to 8 basepairs.

β/β pairing within the hairpin polyamide motif as shown below completelyabolishes DNA-binding:

The present inventor has found that a paired β/β substituted hairpinmotif allows specific targeting of sequences of the form 5′-WGWGWWCW-3′.Substitution of a β/β pair for the second pyrrole pairing of a 12-ringhairpin polyamide, provides polyamides which target a wide variety of 8base pair sequences of mixed sequence composition. Sequences are boundwith subnanomolar affinity and 50-100 fold specificity versus singlebase pair mismatch sites as shown in Table 4.

TABLE 4 Match Mismatch Specificity

8-fold

8-fold

>50-fold

>100-fold

The following examples illustrate particular embodiments of the presentinvention and are not limiting of the specification and claims in anyway.

EXAMPLES Synthesis of Polyamides

A. Materials

Boc-β-alanine-(-4-carboxamidomethyl)-benzyl-ester-copoly(styrene-divinylbenzene)resin (Boc-β-Pam-Resin), dicyclohexylcarbodiimide (DCC),hydroxybenzotriazole (HOBt),2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexa-fluorophosphate (HBTU), Boc-glycine, and Boc-β-alanine werepurchased from Peptides International. N,N-diisopropylethylamine (DIEA),N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), and DMSO/NMPwere purchased from Applied Biosystems. Boc-γ-aminobutyric acid was fromNOVA Biochem, dichloromethane (DCM) and triethylamine (TEA) was reagentgrade from EM, thiophenol (PhSH), dimethylaminopropylamine,trichloroacetyl chloride, N-methylpyrrole, and N-methylimidazole fromAldrich, and trifluoroacetic acid (TFA) from Halocarbon. All reagentswere used without further purification.

¹H NMR were recorded on a GE300 instrument operating at 300 MHz.Chemical shifts are reported in ppm relative to the solvent residualsignal. UV spectra were measured on a Hewlett-Packard Model 8452A diodearray spectrophotometer. IR spectra were recorded on a Perkin-Elmer FTIRspectrometer. High- resolution FAB mass spectra were recorded at theMass Spectroscopy Laboratory at the University of California, Riverside.Matrix- assisted, laser desorption/ionization time of flight massspectrometry was carried out at the Protein and Peptide MicroanalyticalFacility at the California Institute of Technology. HPLC analysis wasperformed either on a HP 1090M analytical HPLC or a Beckman Gold systemusing a RAINEN C₁. Microsorb MV, 5 μm, 300×4.6 mm reversed phase columnin 0.1% (wt/v) TFA with acetonitrile as eluent and a flow rate of 1.0mL/min, gradient elution 1.25% acetonitrile/min. Preparatory HPLC wascarried out on a Beckman HPLC using a Waters DeltaPak 25×100 mm, 100 μm,C₁₈ column equipped with a guard, 0.1% (wt/v) TFA, 0.25%acetonitrile/min. 18MΩ water was obtained from a Millipore MilliQ waterpurification system, and all buffers were 0.2 μm filtered. Thin- layerchromatography (TLC) was performed on silica gel 60 F₂₅₄ precoatedplates. Reagent-grade chemicals were used unless otherwise stated.

B. Synthesis of Boc-Protected Pyrrole and Imidazole Monomer

1. 4-Nitro-2-trichloroacetyl-1-methylpyrrole:

To a well stirred solution of trichloroacetyl chloride (1 kg, 5.5 mole)in 1.5 liter ethyl ether in a 12 liter flask was added dropwise over aperiod of 3 h a solution of N-methylpyrrole (0.45 kg, 5.5 mole) in 1.5liter anhydrous ethyl ether. The reaction was stirred for an additional3 hours and quenched by the dropwise addition of a solution of 400 gpotassium carbonate in 1.5 liters water. The layers were separated andthe ether layer concentrated in vacuo to provide2-(trichloroacetyl)pyrrole (1.2 kg, 5.1 mol) as a yellow crystallinesolid sufficiently pure to be used without further purification. To acooled (−40° C.) solution of 2-(trichloroacetyl) pyrrole (1.2 kg, 5.1mol) in acetic anhydride (6 L) in a 12 L flask equipped with amechanical stirrer was added 440 mL fuming nitric acid over a period of1 hour while maintaining a temperature of (−40° C.). The reaction wascarefully allowed to warm to room temperature and stir an additional 4h. The mixture was cooled to −30° C., and isopropyl alcohol (6 L) added.The solution was stirred at −20° C. for 30 min during which time a whiteprecipitate forms. The solution was allowed to stand for 15 min and theresulting precipitate collected by vacuum filtration to provide4-Nitro-2-trichloroacetyl-1-methylpyrrole. (0.8 kg, 54% yield) TLC (7:2benzene/ethyl acetate) Rf 0.7; ¹H NMR (DMSO-d₆) δ 8.55 (d, 1H, J=1.7Hz), 7.77 (d, 1H, J=1.7 Hz), 3.98 (S, 3 H); ¹³C NMR (DMSO-d₆) δ 173.3,134.7, 133.2, 121.1, 116.9, 95.0, 51.5; IR (KBr) 1694, 1516, 1423, 1314,1183, 1113, 998, 750. FABMS m/e 269.936 (M+H 269.937 calc. forC₇H₅N₂O₃Cl₃).

2. Methyl 4-nitropyrrole-2-carboxylate:

To a solution of 4-Nitro-2-trichloroacetyl-1-methylpyrrole (800 g, 2.9mol) in 2.5 L methanol in a 4 L Erlenmeyer flask equipped with amechanical stirrer was added dropwise a solution of NaH (60% dispersionin oil) (10 g, 0.25 mol) in 500 mL methanol. The reaction was stirred 2h. at room temperature, and quenched by the addition of conc. sulfuricacid (25 mL). The reaction was then heated to reflux, allowed to slowlycool to room temperature as methyl 4-nitropyrrole-2-carboxylatecrystallizes as white needles which were collected by vacuum filtrationand dried in vacuo. (450 g, 47% yield). TLC (ethyl acetate) Rf 0.8; ¹HNMR (DMSO-d₆) δ 8.22 (d, 1H, J=1.7 Hz), 7.22 (d, 1H, J=1.6 Hz), 3.88 (s,3 H), 3.75 (s, 3 H), ¹³C NMR (DMSO-d₆) δ 37.8, 52.2, 112.0, 123.0,129.9, 134.6, 160.3; IR (KBr) 3148, 1718, 1541, 1425, 1317, 1226, 1195,1116, 753. FABMS m/e 184.048 (M+H 184.048 calc. for C₇H₈N₂O₄).

3. Methyl 4-amino-1-methyl-pyrrole-2-carboxylate hydrochloride:

Methyl-4-nitropyrrole-2-carboxylate (450 g, 2.8 mol) was dissolved inethyl acetate (8 L). A slurry of 40 g of 10% Pd/C in 800 mL ethylacetate was then added and the mixture stirred under a slight positivepressure of hydrogen (c.a. 1.1 atm) for 48 h. Pd/C was removed byfiltration through Celite, washed 1×50 mL ethyl acetate, and the volumeof the mixture reduced to c.a. 500 mL. 7 L of cold ethyl ether was addedand HCl gas gently bubbled through the mixture. The precipitated aminehydrochloride was then collected by vacuum filtration to yield (380 g,81.6%) of Methyl 4-amino-1-methyl-pyrrole-2-carboxylate hydrochloride asa white powder. TLC (ethyl acetate) Rf(amine) 0.6, Rf salt (0.0), ¹H NMR(DMSO-d₆) δ 10.23 (br s, 3H), 7.24 (d, 1H J=1.9), 6.79 (d, 1H, J=2.0),3.83 (s, 3H), 3.72 (s, 3H) ¹³C NMR (DMSO-d₆) δ 160.8, 124.3, 121.2,113.4, 112.0, 51.8, 37.1; IR (KBr) 3095, 2693, 1709, 1548, 1448, 1266,1102, 802, 751. FABMS m/e 154.075 (154.074 calc. for C₇H₁₀N₂O₂).

4. 4-[(tert-Butoxycarbonyl)amino]-1-methylpyrrole-2-carboxylic acid:

The hydrochloride salt of the pyrrole amine Methyl4-amino-1-methyl-pyrrole-2-carboxylate hydrochloride (340 g, 1.8 mol)was dissolved in 1 L of 10% aqueous sodium carbonate in a 3 L flaskequipped with a mechanical stirrer, di-t-butyldicarbonate (400 g, 2.0mmol) slurried in 500 mL of dioxane was added over a period of thirtymin maintaining a temperature of 20° C. The reaction was allowed toproceed for three h and was determined complete by TLC, cooled to 5° C.for 2 h and the resulting white precipitate collected by vacuumfiltration. The Boc-pyrrole ester contamination with Boc-anhydride wasdissolved in 700 mL MeOH, 700 mL of 2M NaOH was added and the solutionheated at 60° C. for 6 h. The reaction was cooled to room temperature,washed with ethyl ether (4×1000 mL), the pH of the aqueous layer reducedto c.a. 3 with 10% (v/v) H₂SO₄, and extracted with ethyl acetate (4×2000mL). The combined ethyl acetate extracts were dried (sodium sulfate) andconcentrated in vacuo to provide a tan foam. The foam was dissolved in500 mL of DCM and 2 L petroleum ether added, the resulting slurry wasconcentrated in vacuo. The reaction was redissolved and concentratedthree additional times to provide (320 g, 78% yield) of4-[(tert-Butoxycarbonyl)amino]-1-methylpyrrole-2-carboxylic acid as afine white powder. TLC (7:2 benzene/ethyl acetate v/v) Rf (ester) 0.8,Rf (acid) 0.1. (ethyl acetate), Rf (acid) 0.6, ¹H NMR (DMSO-d₆) δ 12.10(s, 1H), 9.05 (s, 1H), 7.02 (s, 1H), 6.55 (s, 1H), 3.75 (s, 3H), 1.41(s, 9H) ¹³C NMR (DMSO-d₆) δ 162.4, 153.2, 123.3, 120.1, 119.2, 107.9,78.9, 36.6, 28.7.; IR (KBr) 3350, 2978, 1700, 1670, 1586, 1458, 1368,1247, 1112, 887, 779. FABMS m/e 241.119 (M+H 241.119 calc. forC₁₁H₁₇N₂O₄).

5. 1,2,3-Benzotriazol-1-yl4-[(tert-butoxycarbonyl)-amino]-1-methylpyrrole-2-carboxylate:

Boc-Py-acid, 4-[(tert-Butoxycarbonyl)amino]-1-methylpyrrole-2-carboxylicacid (31 g, 129 mmol) was dissolved in 500 mL DMF, HOBt (17.4 g, 129mmol) was added followed by DCC (34 g, 129 mmol). The reaction wasstirred for 24 h and then filtered dropwise into a well stirred solutionof 5 L of ice water. The precipitate was allowed to sit for 15 min at 0°C. and then collected by filtration. The wet cake was dissolved in 500mL DCM, and the organic layer added slowly to a stirred solution of coldpetroleum ether (4° C.). The mixture was allowed to stand at −20° C. for4 h and then collected by vacuum filtration and dried in vacuo toprovide (39 g, 85% yield) of 1,2,3-Benzotriazol-1-yl4-[(tert-butoxycarbonyl)-amino]-1-methylpyrrole-2-carboxylate as afinely divided white powder. TLC (7:2 benzene/ethyl acetate v/v) Rf0.6¹H NMR (DMSO-d₆) δ 9.43 (s, 1H), 8.12 (d, 1H, J=8.4 Hz), 7.80 (d, 1H,J=8.2 Hz), 7.64 (t, 1H, J=7.0 Hz), 7.51 (m, 2H), 7.18 (s, 1H), 3.83 (s,3H), 1.45 (s, 9H), ¹³C NMR (DMSO-d₆) δ 156.5, 153.3, 143.2, 129.6,129.2, 125.7, 125.2, 124.6, 120.3, 112.8, 110.3, 109.8, 79.5, 36.8,28.6.; IR (KBr) 3246, 3095, 2979, 1764, 1711, 1588, 1389, 1365, 1274,1227, 1160, 1101, 999, 824, 748.; FABMS m/e 358.152 (M+H 358.151 calc.for C₁₇H₂₀N₅O₄).

6. Ethyl 1-methylimidazole-2-carboxylate:

N-methylimidazole (320 g, 3.9 mol) was combined with 2 L acetonitrileand 1 L triethylamine in a 12 L flask equipped with a mechanical stirrerand the solution cooled to −20° C. Ethyl chloroformate (1000 g, 9.2 mol)was added with stirring, keeping the temperature between −20° C. and−25° C. The reaction was allowed to slowly warm to room temperature andstir for 36 h. Precipitated triethylamine hydrochloride was removed byfiltration and the solution concentrated in vacuo. at 65° C. Theresulting oil was purified by distillation under reduced pressure (2torr, 102° C.) to provide Ethyl 1-methylimidazole-2-carboxylate as awhite solid (360 g, 82% yield). TLC (7:2 benzene/ethyl acetate v/v) Rf0.2¹H NMR (DMSO-d₆) δ 7.44 (d, 1H, J=2.8 Hz), 7.04 (d, 1H, J=2.8 Hz),4.2 (q, 2 H, J=3.5 Hz), 3.91 (s, 3 H), 1.26 (t, 3 H, J=3.5 Hz); ¹³C NMR(DMSO-d₆) δ 159.3, 129.1, 127.7, 61.0, 36.0, 14.5; IR(KBr) 3403, 3111,2983, 1713, 1480, 1422, 1262, 1134, 1052, 922, 782, 666; FABMS m/e155.083 (M+H 155.083 calc. for C₇H₁₁N₂O₂).

7. Ethyl 1-methyl-4-nitroimidazole-2-carboxylate:

Ethyl 1-methylimidazole-2-carboxylate was carefully dissolved in 1000 mLof concentrated sulfuric acid cooled to 0° C. 90% nitric acid (1 L) wasslowly added maintaining a temperature of 0° C. The reaction was thenrefluxed with an efficient condenser (−20° C.) in a well ventilated hoodfor 50 min. The reaction was cooled with an ice bath, and quenched bypouring onto 10 L ice. The resulting blue solution was then extractedwith 20 L DCM, the combined extracts dried (sodium sulfate) andconcentrated in vacuo to yield a tan solid which was recrystallized from22 L of 21:1 carbon tetrachloride/ethanol. The resulting white crystalsare collected by vacuum filtration to provide pure Ethyl1-methyl-4-nitroimidazole-2-carboxylate. (103 g, 22% yield). TLC (7:2benzene/ethyl acetate v/v) Rf 0.5, ¹H NMR (DMSO-d₆) δ 8.61 (s, 1H), 4.33(q, 2 H, J=6.4 Hz), 3.97 (s, 3 H), 1.29 (t, 3 H, J=6.0 Hz), ¹³C NMR(DMSO-d₆) δ 158.2, 145.4, 135.3, 127.4, 62.2, 37.3, 14.5; IR (KBr) 3139,1719, 1541, 1498, 1381, 1310, 1260, 1122, 995, 860, 656.; FABMS m/e200.066 (M+H 200.067 calc. for C₇H₁₀N₃O₄).

8. Ethyl 4-amino-1-methylimidazole-2-carboxylate hydrochloride:

The nitro imidazole ethyl ester Ethyl1-methyl-4-nitroimidazole-2-carboxylate (103 g, 520 mmol) was dissolvedin 5 L of 1:1 ethanol/ethyl acetate, 20 g 10% Pd/C slurried in 500 mLethyl acetate was added and the mixture stirred under a slight positivepressure of hydrogen (c.a. 1.1 atm) for 48 h. The reaction mixture wasfiltered, concentrated in vacuo to a volume of 500 mL and 5 L of coldanhydrous ethyl ether added. Addition of HCl gas provided a whiteprecipitate. The solution was cooled at −20° C. for 4 h and theprecipitate collected by vacuum filtration and dried in vacuo to provide(75 g, 78% yield) of ethyl 4-amino-1-methylimidazole-2-carboxylatehydrochloride as a fine white powder. TLC (7:2 benzene: ethyl acetate)R_(f) (amine) 0.3, R_(f) (salt) 0.0. ¹H NMR (DMSO-d₆) δ 10.11 (br, s,3H), 7.43 (s, 1H), 4.28 (q, 2H, J=7.1 Hz), 3.92 (s, 1H), 1.28 (t, 3H,J=7.1 Hz) ¹³C NMR (DMSO-d₆) δ 157.6, 132.6, 117.4, 117.3, 61.8, 36.6,14.5; IR (KBr) 3138, 2883, 1707, 1655, 1492, 1420, 1314, 1255, 1152,1057, 837, 776.; FABMS m/e 169.085 (169.084 calc. for C₇H₁₁N₃O₂).

9. 4-[(tert-butoxycarbonyl)amino]-1-methylimidazole-2-carboxylic acid:

The imidazole amine ethyl 4-amino-1-methylimidazole-2-carboxylatehydrochloride (75 g, 395 mmol) was dissolved in 200 mL DMF. DIEA (45 L,491 mmol) was added followed by di-t-butyldicarbonate (99 g, 491 mmol).The mixture was shaken at 60° C. for 18 h, allowed to assume roomtemperature, and partitioned between 500 mL brine, 500 mL ethyl ether.The ether layer was extracted (2×200 mL each) 10% citric acid, brine,satd. sodium bicarbonate, brine, dried over sodium sulfate andconcentrated in vacuo to yield the Boc-ester contaminated with 20%Boc-anhydride as indicated by ¹H NMR. The Boc-ester, used withoutfurther purification, was dissolved in 200 mL 1 M NaOH. The reactionmixture was allowed to stand for 3 h at 60° C. with occasionalagitation. The reaction mixture was cooled to 0° C., and carefullyneutralized with 1 M HCl to pH 2, at which time a white gel forms. Thegel was collected by vacuum filtration, frozen before drying, andremaining water lyophilized to yield4-[(tert-butoxycarbonyl)amino]-1-methylimidazole-2-carboxylic acid as awhite powder. (51 g, 54% yield). ¹H NMR (DMSO-d₆) δ 9.47 (s, 1H), 7.13(s, 1H), 3.85 (s, 3H), 1.41 (s, 9H). ¹³C NMR (DMSO-d₆) δ 160.9, 152.9,137.5, 134.5, 112.4, 79.5, 35.7, 28.6; IR(KBr) 3448, 2982, 1734, 1654,1638, 1578, 1357, 1321, 1249, 1163, 799.; FABMS m/e 241.105 (241.106calc. for C₁₀H₁₅N₃O₄).

10. γ-[(tert-butoxycarbonyl)amino]-butyric acid-(4-carboxamido-1-methyl-imidazole)-2-carboxylic acid:

To a solution of Boc-γ-aminobutyric acid (10 g, 49 mmol) in 40 mL DMFwas added 1.2 eq HOBt (7.9 g, 59 mmol) followed by 1.2 eg DCC (11.9 g,59 mmol). The solution was stirred for 24 h, and the DCU removed byfiltration. Separately, to a solution of ethyl4-nitro-1-methylimidazole-2-carboxylate (9.8 g, 49 mmol) in 20 mL DMFwas added Pd/C catalyst (10%, 1 g), and the mixture was hydrogenated ina Parr bom apparatus (500 psi H₂) for 2 h. The catalyst was removed byfiltration through celite and filtrate immediately added to the −OBtester solution. An excess of DIEA (15 mL) was then added and thereaction stirred at 37° C. for 48 h. The reaction mixture was then addeddropwise to a stirred solution of ice water and the resultingprecipitate collected by vacuum filtration to provide crude ethylγ-[[(tert-butoxy)carbonyl]amino]-butyric acid-(4-carboxamido-1-methyl-pyrrole)-2-carboxylate (5 g, 14.1 mmol). To thecrude ester dissolved in 50 mL methanol was added 50 mL 1M KOH and theresulting mixture allowed to stir for 6 h at 37° C. Excess methanol wasremoved in vacuo and the resulting solution acidified by the addition of1 m HCl. The resulting precipitate was collected by vacuum filtrationand dried in vacuo to yield γ-[(tert-butoxycarbonyl)amino]butyric acid-(4-carboxamido-1-methyl-imidazole)-2-carboxylic acid as a brown powder.(4.4 g, 89% yield), ¹H NMR (DMSO-d₆) δ 10.50 (s, 1H), 7.45 (s, 1H), 6.82(t, 1H, J=3.6 Hz), 3.86 (s, 3 H), 2.86 (q, 2 H, J=4.6 Hz), 2.22 (t, 2 H,J=7.4 Hz), 1.57 (qunitet, 2 H, J=5.9 Hz), 1.29 (s, 9 H); IR 3416, 2950,2841, 1650,1538 1449, 1392, 1250, 1165, 1108; FABMS m/e 326.160 (326.159calc. for C₁₄H₂₂N₄O₅).

11.4-[(tert-butoxycarbonyl)amino]-1-methylpyrrole-2-(4-carboxamido-1-methylimidazole)-2-carboxylic acid:

4-[(tert-butoxycarbonyl)amino]-1-methylpyrrole-2-(4-carboxamido-1-methylimidazole)-2-carboxylic acid was prepared as described below forγ-[(tert-butoxycarbonyl)-amino]-butyric acid-(4-carboxamido-1-methyl-imidazole)-2-caroboxylic acid substitutingBoc-Pyrrole acid for Boc-γ-aminobutyric acid. (4.1 g, 91% yield). ¹H NMR(DMSO-d₆) δ 10.58 (s, 1H), 9.08 (S, 1H), 7.57 (s, 1H), 6.97 (s, 1 H),6.89 (S, 1H), 3.89 (s, 3 H), 3.75 (s, 3 H), 1.35 (s, 9 H); ¹³C NMR(DMSO-d₆) δ 160.36, 159.1, 153.4, 137.9, 132.3, 122.8, 122.3, 118.5,115.5, 105.5, 105.4, 78.8, 28.7, 24.9; IR 3346, 2929, 1685, 1618, 1529,1342, 1274, 1179, 997, 761. FABMS m/e 364.161 (364.162 calc. forC₁₆H₂₂N₅O₅).

C. Solution Phase Synthesis of Polyamides Using Boc-Protected Pyrroleand Imidazole Building Blocks.

1. Aminohexa-(N-methylpyrrolecarboxamide) ditrifluoracetate:

To a solution of N-(tert-butoxycarbonyl)-tris(N-methylpyrrolecarboxamide) (20 mg, 41 μmol) in DMF (100 μl) was addedHBTU (26 mg, 69 μmol) followed by DIEA (50 μl, 288 μmol). The reactionwas allowed to stand for 5 minutes, agitated, and allowed to stand foran additional five minutes. Aminotris-(N-methylpyrrolecarboxamide) (24mg, 41 μmol) was then added followed by DIEA (50 μl, 288 μmol) and thereaction agitated for 2 hours. The reaction mixture was concentrated invacuo and TFA (10 ml) added. After 2 minutes the TFA was removed invacuo. Purification of the resulting brown oil by reversed phase HPLCafforded the diamine aminohexa-(N-methylpyrrolecarboxamide)ditrifluoroacetate as a white powder. Yield: 26 mg (58%); ¹H NMR(DMSOd₆) δ 10.06 (s, 1 H), 9.95 (m, 2 H), 9.91 (s, 1 H), 9.84 (s, 1 H),9.44 (br s, 1 H), 8.16 (t, 1 H, J=4.0 Hz), 7.22 (m, 4 H), 7.16 (d, 1 H,J=1.7 Hz), 7.10 (s, 1 H, J=1.7 Hz), 7.07 (m, 3 H), 6.98 (s, 1 H, J=1.7Hz), 6.93 (s, 1 H J=1.8 Hz), 3.88 (m, 6 H), 3.84 (m, 12 H), 3.79 (m, 6H), 3.21 (m, 2 H), 3.04 (m, 2 H), 2.77 (d, 6 H, J=4.8 Hz), 1.80 (m, 2H); FABMS m/e 835.412 (M+H, 835.416 calc. for C₄₁H₅₁N₁₄O₆).

2. ImPyPyPyPyPyPy-Dp:

N-methyl-Imidazole-2-carboxylic acid (100 mg, 741 μmol) and HOBt (72 mg,500 μmol) were suspended in 500 μl DMF. Upon addition of DCC (100 mg,500 μmol) the reaction mixture become a homogeneous solution. Theactivation was allowed to stand for 12 hours, precipitateddicyclohexylurea removed by filtration andAminohexa-(N-methylpyrrolecarboxamide) ditrifluoroacetate (10 mg, 9.4μmol) added followed by DIEA (100 μl, 576 μmol), and the reactionallowed to stand for 2 hours. Reversed phase HPLC purification of thereaction mixture afforded ImPyPyPyPyPyPy-Dp as a white powder. Yield:6.3 mg (62%); HPLC, r.t. 27.4 min; UV λ_(max) (ε), 246 (34,100), 304(56,600) nm; ¹H NMR (DMSO-d₆) δ 10.46 (s, 1 H), 9.55 (s, 1 H), 9.94 (m,3 H), 9.90 (s, 1 H), 9.20 (br s, 1 H), 8.14 (t, 1 H, J=7.2 Hz), 7.38 (s,1 H), 7.28 (d, 1 H, J=1.4 Hz), 7.26 (d, 1 H, J=1.4 Hz), 7.23 (m, 4 H),7.08 (m, 5 H), 7.04 (s, 1 H, J=1.2 Hz), 6.93 (d, 1 H, J=1.6 Hz), 3.98(s, 3 H), 3.84 (m, 15 H), 3.83 (s, 1 H), 3.30 (q, 2 H, J=7.4 Hz), 3.21(t, 2 H, J=7.1 Hz), 2.77 (d, 6H, J=4.1 Hz), 1.74 (m, 2 H); MALDI-TOF MS944.21 (M+H 944.04 calc.); FABMS m/e 965.430 (M+Na, 965.426 calc. forC₄₆H₅₄N₁₆O₇Na).

D. Solid Phase Syntheses

1. Activation of Imidazole-2-carboxylic acid, Boc-γ-aminobutyric acid,Boc-glycine, and Boc-β-alanine

The appropriate amino acid or acid (2 mmol) was dissolved in 2 mL DMF.HBTU (720 mg, 1.9 mmol) was added followed by DIEA (1 mL) and thesolution lightly shaken for at least 5 min.

2. Activation of Boc-Imidazole acid

Boc imidazole acid (257 mg, 1 mmol) and HOBt (135 mg, 1 mmol) weredissolved in 2 mL DMF, DCC (202 mg, 1 mmol) is then added and thesolution allowed to stand for at least 5 min.

3. Activation of Boc-γ-Imidazole acid and Boc-Pyrrole-Imidazole acid:

The appropriate dimer (1 mmol) and HBTU (378 mg, 1 mmol) are combined in2 mL DMF. DIEA (1 mL) is then added and the reaction mixture allowed tostand for 5 min.

4. Activation of Boc-Pyrrole acid. (for coupling to Imidazole amine)

Boc-Pyrrole acid (514 mg, 2 mmol) was dissolved in 2 mL dichloromethane,DCC (420 mg, 2 mmol) added, and the solution allowed to stand for 10min. DMAP (101 mg, 1 mmol) was added and the solution allowed to standfor 1 min.

5. Acetylation Mix:

2 mL DMF, DIEA (710 μL, 4.0 mmol), and acetic anhydride (380 μL, 4.0mmol) were combined immediately before use.

6. Manual Synthesis Protocol:

Bocβ-alanine-Pam-Resin (1.25 g, 0.25 mmol) is placed in a 20 mL glassreaction vessel, shaken in DMF for 5 min and the reaction vesseldrained. The resin was washed with DCM (2=30 s.) and the Boc groupremoved with 80% TFA/DCM/0.5M PhSH, 1=30s., 1=20 min The resin waswashed washed with DCM (2=30 s.) followed by DMF (1=30 s.) A resinsample (5-10 mg) was taken for analysis. The vessel was drainedcompletely and activated monomer added, followed by DIEA if necessary.The reaction vessel was shaken vigorously to amke a slurry. The couplingwas allowed to proceed for 45 min, and a resin sample taken. Thereaction vessel was then washed with DCM, followed by DMF.

7. Machine-Assisted Protocols:

Machine-assisted synthesis was performed on a ABI 430A synthesizer on a0.18 mmol scale (900 mg resin; 0.2 mmol/gram). Each cycle of amino acidaddition involved: deprotection with approximately 80% TFA/DCM/0.4M PhSHfor 3 minutes, draining the reaction vessel, and then deprotection for17 minutes; 2 dichloromethane flow washes; an NMP flow wash; drainingthe reaction vessel; coupling for 1 hour with in situ neutralization,addition of dimethyl sulfoxide (DMSO)/NMP, coupling for 30 minutes,addition of DIEA, coupling for 30 minutes; draining the reaction vessel;washing with DCM, taking a resin sample for evaluation of the progressof the synthesis by HPLC analysis; capping with acetic anhydride/DIEA inDCM for 6 minutes; and washing with DCM. A double couple cycle isemployed when coupling aliphatic amino acids to imidazole, all othercouplings are performed with single couple cycles.

The ABI 430A synthesizer was left in the standard hardware configurationfor NMP-HOBt protocols. Reagent positions 1 and 7 were DIEA, reagentposition 2 was TFA/0.5M thiophenol, reagent position 3 was 70%ethanolamine/methanol, regent position 4 was acetic anhydride, reagentposition 5 was DMSO/NMP, reagent position 6 was methanol, and reagentposition 8 was DMF. New activator functions were written, one for directtransfer of the cartridge contents to the concentrator (switch list 21,25, 26, 35, 37, 44), and a second for transfer of reagent position 8directly to the cartridge (switch list 37, 39, 45, 46).

Boc-Py-OBt ester (357 mg, 1 mmol) was dissolved in 2 ml DMF and filteredinto a synthesis cartridge. Boc-Im acid monomer was activated(DCC/HOBt), filtered, and placed in a synthesis cartridge.Imidazole-2-carboxylic acid was added manually. At the initiation of thecoupling cycle the synthesis was interrupted, the reaction vessel ventedand the activated monomer added directly to the reaction vessel throughthe resin sampling loop via syringe. When manual addition was necessaryan empty synthesis cartridge was used. Aliphatic amino acids (2 mmol)and HBTU (1.9 mmol) were placed in a synthesis cartridge. 3 ml of DMFwas added using a calibrated delivery loop from reagent bottle 8,followed by calibrated delivery of 1 ml DIEA from reagent bottle 7, anda 3 minute mixing of the cartridge.

The activator cycle was written to transfer activated monomer directlyfrom the cartridge to the concentrator vessel, bypassing the activatorvessel. After transfer, 1 ml of DIEA was measured into the cartridgeusing a calibrated delivery loop, and the DIEA solution combined withthe activated monomer solution in the concentrator vessel. The activatedester in 2:1 DMF/DIEA was then transferred to the reaction vessel. Alllines were emptied with argon before and after solution transfers.

8. Stepwise HPLC analysis:

A resin sample (c.a. 4 mg) was placed in a 4 mL glass test tube, 200 μLof N,N-dimethylaminopropylamine was added and the mixture heated at 100°C. for 5 min. The cleavage mixture was filtered and a 25 μL sampleanalyzed by analytical HPLC at 254 nm.

9. Typical Manual Synthesis Protocol: PyPyPy-γ-ImImPy-β-Dp:

Boc-β-Pam-resin (1.25 g, 0.25 mmol amine) was shaken in DMF for 30 minand drained. The N-Boc group removed by washing with DCM for 2=30 s.followed by a 1 min shake in 80% TFA/DCM/0.5M PhSH, draining thereaction vessel and a brief 80% TFA/DCM/0.5 M PhSH wash, and 20 minshaking in 80% TFA/DCM/0.5M PhSH solution. The resin was washed 1 minwith DCM and 30 s with DMF. A resin sample (8-10 mg) was taken foranalysis. The resin was drained completely and Boc-pyrrole-Obt monomer(357 mg, 1 mmol) dissolved in 2 ml DMF added followed by DIEA (1 ml) andthe resin shaken vigorously to make a slurry. The coupling was allowedto proceed for 45 min. A resin sample (8-10 mg) was taken after 40 minto check reaction progress. The reaction vessel was washed with DMF for30 s and dichloromethane for 1 min to complete a single reaction cycle.Six additional cycles were performed adding, BocIm-OH (DCC/HOBt),BocIm-OH (DCC/HOBt), Boc-γ-aminobutyric acid (HBTU/DIEA) and allowed tocouple for 2 hours, BocPy-OBt, BocPy-OBt, and pyrrole-2-carboxylic acid(HBTU/DIEA). The resin was washed with DMF, DCM, MeOH, and ethyl etherand then dried in vacuo. PyPyPy-γ-ImImPy-β-Pam-Resin (180 mg, 29 μmol)¹²was weighed into a glass scintillation vial, 1.5 ml ofN,N-dimethylaminopropylamine added, and the mixture heated at 55° C. for18 hours. The resin was removed by filtration through a disposablepolyproplene filter and washed with 5 ml of water, the amine solutionand the water washes combined, and the solution loaded on a C₁₈preparatory HPLC column, the column allowed to wash for 4 min in 0.1%TFA at 8 ml/min, the polyamide was then eluted in 100 min. as a welldefined peak with a gradient of 0.25% acetonitrile per min. Thepolyamide was collected in four separate 8 ml fractions, the purity ofthe individual fractions verified by HPLC and ¹H NMR, to providepurified PyPyPy-γ-ImImPy-β-Dp: (11.2 mg, 39% recovery), UV λ_(max), 246(31,100), 312 (51,200) HPLC, r.t. 23.6, ¹H NMR (DMSO-d₆) δ 10.30 (s, 1H), 10.26 (s, 1 H), 9.88 (s, 1 H), 9.80 (s, 1 H), 9.30 (s, 1 H), 9.2 (brs, 1 H), 8.01 (m, 3 H), 7.82 (br s 1 H), 7.54 (s, 1 H), 7.52 (s, 1 H),7.20 (d, 1 H, J=1.3 Hz), 7.18 (d, 1 H, J=1.2 Hz), 7.15 (d, 1 H, J=1.3Hz), 7.01 (d, 1 H, J=1.4 Hz), 6.96 (d, 1 H, J=1.4 Hz), 6.92 (d, 1 H,J=1.8 Hz), 6.89 (m, 2 H), 6.03 (t, 1 H, J=2.4 Hz), 3.97 (s, 3 H), 3.96(s, 3 H), 3.85 (s, 3 H), 3.82 (s, 3 H), 3.78 (m, 6 H), 3.37 (m, 2 H),3.20 (q, 2 H J=5.7 Hz), 3.08 (q, 2 H J=6.6 Hz), 2.94 (q, 2 H J=5.3 Hz),2.71 (d, 6 H J=5.8 Hz), 2.32 (m, 4 H), 1.83 (m, 4 H); MALDI-TOF-MS,978.7 (979.1 calc. for M+H).

9. ImImPy-γ-PyPyPy-β-Dp:

Polyamide was prepared by machine assisted solid phase synthesisprotocols and 900 mg resin cleaved and purifed to provideImImPy-γ-PyPyPy-β-Dp as a white powder. (69 mg, 48% recovery), UVλ_(max), 246 (43,300), 308 (54,200) HPLC, r.t. 23.9, ¹H NMR (DMSO-d₆) δ10.31 (s, 1 H), 9.91 (s, 1 H), 9.90 (s, 1 H), 9.85 (s, 1 H), 9.75 (s, 1H), 9.34 (br s, 1 H), 8.03 (m, 3 H), 7.56 (s, 1 H), 7.46 (s, 1 H), 7.21(m, 2 H), 7.15 (m, 2 H), 7.07 (d, 1 H J=1.2 Hz), 7.03 (d, 1 H, J=1.3Hz), 6.98 (d, 1 H, J=1.2 Hz), 6.87 (m, 2 H), 4.02 (m, 6 H), 3.96 (m, 6H), 3.87 (m, 6 H), 3.75 (q, 2 H, J=4.9 Hz), 3.36 (q, 2 H, J=4.0 Hz),3.20 (q, 2 H, J=4.7 Hz), 3.01 (q, 2 H J=5.1 Hz), 2.71 (d, 6H, J=4.8 Hz),2.42 (m, 4 H), 1.80 (m, 4 H) MALDI-TOF-MS 978.8, (979.1 calc. for M+H)

10. AcImImPy-γ-PyPyPy-β-Dp:

Polyamide was prepared by manual solid phase protocols and isolated as awhite powder. (8 mg, 28% recovery), UV λ_(max), 246 (43,400), 312(50,200) HPLC, r.t. 24.8, ¹H NMR (DMSO-d₆) δ 10.35 (s, 1 H), 10.30 (s, 1H), 9.97 (s, 1 H), 9.90 (s, 1 H), 9.82 (s, 1 H), 9.30 (s, 1 H), 9.2 (brs, 1H), 8.02 (m, 3 H), 7.52 (s, 1 H). 7.48 (s, 1 H), 7.21 (m, 2H), 7.16(d, 1 H, J=1.1 Hz), 7.11 (d, 1 H, J=1.2 Hz), 7.04 (d, 1 H, J=1.1 Hz),6.97 (d, 1 H, J=1.3 Hz), 6.92 (d, 1 H, J=1.4 Hz), 6.87 (d, 1 H, J=1.2Hz), 3.99 (s, 3 H), 3.97 (s, 3 H), 3.83 (s, 3 H), 3.82 (s, 3 H), 3.80(s, 3 H), 3.79 (s, 3 H), 3.47 (q, 2 H, J=4.7 Hz), 3.30 (q, 2 H, J=4.6Hz), 3.20 (q, 2 H, J=5.0 Hz), 3.05 (q, 2 H, J=5.1 Hz), 2.75 (d, 6 H,J=4.1 Hz), 2.27 (m, 4 H), 2.03 (s, 3 H), 1.74 (m, 4 H) MALDI-TOF-MS,1036.4 (1036.1 calc. for M+H).

11. AcPyPyPy-γ-ImImPy-β-Dp:

Polyamide was prepared by machine assisted solid phase methods protocolsas a white powder. (14 mg, 48% recovery), UV λ_(max), 246 (44,400), 312(52,300) HPLC, r.t. 23.8, ¹H NMR (DMSO-d₆) 10.32 (s, 1 H), 10.28 (s, 1H), 9.89 (m, 2 H), 9.82 (s, 1 H), 9.18 (s, 1 H), 9.10 (br s, 1 H), 8.03(m, 3 H), 7.55 (s, 1 H), 7.52 (s, 1 H), 7.21 (d, 1 H, J=1.1 Hz), 7.18(d, 1 H, J=7.16 ), 7.15 (d, 1 H, J=1.0 Hz), 7.12 (d, 1 H, J=1.0 Hz),7.02 (d, 1 H, J=1.0 Hz), 6.92 (d, 1 H, J=1.1 Hz), 6.87 (d, 1H, J=1.1Hz), 6.84 (d, 1H, J=1.0 Hz), 3.97 (s, 3 H), 3.93 (s, 3 H), 3.87 (s, 3H), 3.80 (s, 3 H), 3.78 (m, 6 H), 3.35 (q, 2 H, J=5.6 Hz), 3.19 (q, 2 H,J=5.3 Hz), 3.08 (q, 2 H, J=5.7 Hz), 2.87 (q, 2 H, J=5.8 Hz), 2.71 (d, 6H, J=4.0 Hz), 2.33 (m, 4 H), 1.99 (s, 3 H), 1.74 (m, 4 H). MALDI-TOF-MS,1036.2 (1036.1 calc. for M+H).

12. ImPyPy-γ-PyPyPy-β-Dp:

ImPyPy-γ-PyPyPy-β-Pam-Resin was prepared by machine-assisted synthesisprotocols. A sample of resin (1 g, 0.17 mmol was placed in a 20 mL glassscintillation vial, 4 mL of dimethylaminopropylamine added, and thesolution heated at 55° C. for 18 h. Resin substitution is calculated asL_(new)(mmol/g)=L_(old)/(1+L_(old) (W_(new−)W_(old))=10⁻³); L is theloading, and W is the molecular weight of the polyamide attached to theresin. (Barlos, et al. Int. J. Peptide Protein Res. 1991, 37, 513.)Resin is removed by filtration through a disposable proplyene filter and16 mL of water added. The polyamide/amine mixture was purified directlyby preparatory HPLC and the appropriate fractions lyophylized to yield awhite powder. (103 mg, 61% recovery) HPLC r.t. 24.1, UV λ_(max)(H₂O)(ε), 234 nm (39,300), 304 nm (52,000); ¹H NMR (DMSO-d₆); 10.47 (s, 1 H),9.91 (s, 1 H), 9.89 (s, 1 H), 9.87 (s, 1 H), 9.84 (s, 1 H), 9.2 (br s, 1H), 8.08 (m, 3 H), 7.38 (s, 1 H), 7.26 (d, 1 H, J=1.0 Hz), 7.20 (d, 1 H,J=1.0 Hz), 7.14 (m, 4 H), 7.04 (d, 1 H, J=1.1 Hz), 7.02 (d, 1 H, J=1.1Hz), 6.89 (d, 1 H, J=1.0 Hz), 6.85 (m, 2 H), 3.97 (s, 3 H), 3.82 (m, 6H), 3.81 (s, 3 H), 3.77 (m, 6 H), 3.34 (m, 2 H, J=3.9 Hz), 3.18 (m, 2 H,J=5.5 Hz), 3.06 (m, 2 H, J=5.7 Hz), 2.95 (m, 2 H, J=4.9 Hz), 2.71 (d, 6H, J=4.6 Hz), 2.30 (m, 6 H), 1.75 (m, 4 H); MALDI-TOF MS 978.0 (978.1calc. for M+H).

13. ImPyPy-γ-PyPyPy-G-Dp:

ImPyPy-γ-PyPyPy-G-Dp was prepared as described for ImPyPy-γ-PyPyPy-γ-Dp.(12 mg, 40% recovery). HPLC, r.t. 26.9, UV λ_(max) (H₂O), 246 (41,100),306 (51,300) ¹H NMR (DMSO-d₆) δ 10.50 (s, 1 H), 9.95 (s, 1 H), 9.93 (s,1 H), 9.92 (s, 1 H), 9.86 (s, 1 H), 9.2 (br s, 1H), 8.29 (t, 1 H, J=4.4Hz), 8.07 (t, 1 H, J=5.2 Hz), 8.03 (t, 1 H, J=5.4 Hz), 7.39 (s, 1 H),7.27, (d, 1 H, J=1.6 Hz), 7.22 (m, 2 H), 7.16 (m, 2 H), 7.04 (m, 2 H),6.92 (d, 1 H, J=1.6 Hz), 6.89 (d, 1 H, J=1.7 Hz), 6.86 (d, 1 H, J=1.6Hz), 3.97 (s, 3 H), 3.82 (m, 6 H), 3.81 (s, 3 H), 3.78 (m, 6 H), 3.70(d, 2 H, J=5.7 Hz), 3.20 (q, 2 H, J=5.7), 3.11 (q, 2 H, J=4.2 Hz), 3.00(q, 2 H, J=4.4 Hz), 2.76 (d, 6 H, J=4.7 Hz), 2.24 (t, 2 H, J=4.8 Hz),1.77 (m, 4 H); MALDI-TOF-MS, 964.3 (964.1 calc. for M+H).

14. AcImPyPy-γ-PyPyPy-G-Dp:

AcImPyPy-γ-PyPyPy-G-Dp was prepared as described forImPyPy-γ-PyPyPy-β-Dp. (13.1 mg, 30% yield) HPLC, r.t. 24.0, UV λ_(max)(H₂O), 246 (35,900), 312 (48,800) ¹H NMR (DMSO-d₆) δ 10.23 (s, 1 H),9.98 (s, 1 H), 9.32 (s, 1 H), 9.90 (m, 2 H), 9.84 (s, 1 H), 9.2 (br s, 1H), 8.27 (t, 1 H, J=5.0), 8.05 (m, 2 H), 7.41 (s, 1 H), 7.25 (d, 1 H,J=1.4 Hz), 7.22 (m, 2 H), 7.16 (m, 2 H), 7.12 (d, 1 H, J=1.7 Hz), 7.05(d, 1 H, J=1.5 Hz), 6.94 (d, 1 H, J=1.6 Hz), 6.89 (d, 1 H, J=1.7 Hz)6.87 (d, 1 H, J=1.6 Hz), 3.93 (s, 3 H), 3.83 (s, 3 H), 3.82 (m, 6 H),3.81 (s, 3 H), 3.79 (s, 3 H), 3.71 (d, 2 H, J=5.1 Hz), 3.19 (q, 2 H,J=5.8 Hz), 3.12 (q, 2 H, J=5.0 Hz), 3.01 (q, 2 H, J=4.2 Hz), 2.74 (d, 6H, J=4.6 Hz), 2.26 (t 2 H, J=4.6 Hz), 2.00 (s, 3 H), 1.75 (m, 4 H);MALDI-TOF-MS, 1021.6 (1021.1 calc. for M+H).

15. AcImPyPy-γ-PyPyPy-β-Dp:

AcImPyPy-γ-PyPyPy-β-Dp was prepared as described forImPyPy-γ-PyPyPy-β-Dp. (9.2 mg, 31% yield) UV λ_(max) (H₂O), 246(42,800), 312 (50,400) HPLC, r.t. 24.9, ¹H NMR (DMSO-d₆) δ 10.25 (s, 1H), 10.01 (s, 1 H, 9.92 (m, 3 H), 9.86 (s, 1 H), 9.3 (br s, 1 H), 8.10(m, 3 H), 7.42 (s, 1 H), 7.25 (d, 1 H, J=1.5 Hz), 7.20 (d, 1 H, J=1.6Hz), 7.16 (m, 3 H), 7.12 (d, 1 H, J=1.4 Hz), 7.03 (d, 1 H, J=1.7), 6.89(d, 1 H, J=1.6 Hz), 6.86 (m, 2 H), 3.92 (s, 3 H), 3.83 (s, 3 H), 3.82(s, 3 H), 3.80 (s, 6H), 3.78 (s, 3 H), 3.35 (q, 2 H, J=5.5 Hz), 3.20 (q,2 H, J=3.8 Hz), 3.08 (q, 2 H, J=3.3 Hz), 2.97 (q, 2 H, J=3.8 Hz), 2.75(d, 6 H, J=4.8 Hz), 2.34 (t, 2 H, J=5.0 Hz), 2.24 (t, 2 H, J=4.4 Hz),2.00 (s, 3 H), 1.71 (m, 4 H); MALDI-TOF-MS, 1035.4 (1035.1 calc. forM+H).

16. ImImIm-γ-PyPyPy-β-Dp.

The product was synthesized by manual solid phase protocols andrecovered as a white powder (2.4 mg, 4% recovery). UV λ_(max) 312(48,500); ¹H NMR (DMSO-d₆) d 10.09 (s, 1 H), 9.89 (s, 1 H), 9.88 (s, 1H), 9.83 (s,1 H), 9.57 (s, 1 H), 9.19 (br s, 1 H), 8.36 (t, 1 H, J=5.6Hz), 8.03 (m, 2 H), 7.64 (s, 1 H), 7.51 (s, 1 H), 7.45 (s, 1 H), 7.20(d, 1 H, J=1.0 Hz), 7.15 (d, 1 H, J=2.0 Hz), 7.14 (s, 1 H), 7.08 (s, 1H), 7.0 (s, 1 H), 6.87 (d, 2 H, J=2.2 Hz), 4.01 (s, 3 H), 3.99 (s, 3 H),3.95 (s, 3 H), 3.82 (s, 3 H), 3.82 (s, 3 H), 3.79 (s, 3 H), 3.37 (q, 2H, J=5.8 Hz), 3.26 (q, 2 H, J=6.1 Hz), 3.10 (q, 2 H, J=6.1 Hz), 2.99 (m,2 H), 2.73 (d, 6 H, J=4.8 Hz), 2.34 (t, 2 H, J=7.2 Hz), 2.27 (t, 2 H,J=7.3 Hz), 1.79 (m, 4 H); MALDI-TOF-MS, 980.1 (980.1 calc. for M+H).

17. ImImIm-γ-PyPyPy-β-Dp-NH₂:

A sample of machine-synthesized resin (350 mg, 0.17 mmol/gram¹) wasplaced in a 20 mL glass scintillation vial, and treated with 2 mL3,3′-diamino-N-methyldipropylamine at 55° C. for 18 hours.

The resin was removed by filtration through a disposable propylenefilter, and the resulting solution dissolved with water to a totalvolume of 8 mL, and purified directly by preparatory reversed phase HPLCto provide ImImIm-γ-PyPyPy-β-Dp-NH₂ (28 mg, 41% recovery) as a whitepowder. ¹H NMR (DMSO-d₆) δ 10.14 (s, 1 H), 9.89 (s, 1 H), 9.88 (s, 1 H),9.83 (s, 1 H), 9.6 (br s, 1 H), 9.59 (s, 1 H), 8.36 (t, 1 H, J=5.5 Hz),8.09 (t, 1 H, J=5.0 Hz), 8.03 (t, 1 H, J=5.0 Hz), 7.9 (br s, 3 H), 7.63(s, 1 H), 7.50 (s, 1 H), 7.44 (s, 1 H), 7.19 (d, 1 H, J=1.2 Hz), 7.13(m, 2 H), 7.08 (d, 1 H, J=1.3 Hz), 7.02 (d, 1 H, J=1.2 Hz), 6.85 (m, 2H), 4.01 (s, 3 H), 3.99 (s, 3 H), 3.97 (m, 6 H), 3.80 (s, 3 H), 3.77 (s,3 H), 3.44 (q, 2 H, J=5.3 Hz), 3.23 (q, 2 H, J=6.0 Hz), 3.05 (m, 6 H),2.83 (q, 2 H, J=5.0 Hz), 2.70 (d, 3 H, J=4.0 Hz), 2.32 (t, 2 H, J=6.9Hz), 2.25 (t, 2 H, J=6.9 Hz), 1.90 (m, 2 H), 1.77 (m, 4 H).MALDI-TOF-MS, 1022.8 (1023.1 calc. for M+H).

18. ImPyPy-G-PyPyPy-G-Dp-NH₂:

Polyamide was prepared by manual solid phase methods as a white powderupon cleavage of 240 mg resin with N-methyl-bis(aminopropyl)amine (2 ml,55° C.) (19.0 mg, 44% recovery after HPLC purification). ¹H NMR(DMSO-d₆) δ 10.49 (s, 1 H), 9.97 (s, 1 H), 9.93 (s, 1 H), 9.91 (s, 1 H),9.89 (s, 1 H), 9.7 (br s, 1 H), 8.27 (m, 2 H), 8.04 (t, 1 H, J=5.1 Hz),7.88 (br s, 3 H), 7.39 (s, 1 H), 7.27 (d, 1 H, J=1.6 Hz), 7.21 (m, 3 H),7.15 (m, 2 H), 7.05 (m, 2 H), 6.93 (m, 3 H), 3.97 (s, 3 H), 3.96 (m, 6H), 3.92 (m, 9 H), 3.72 (m, 4 H), 3.14 (m, 6 H), 3.05 (q, 2 H, J=5.4Hz), 2.73 (d, 3 H, J=3.3 Hz), 1.88 (quintet, 2 H, J=4.6 Hz), 1.75(quintet, 2 H, J=6.3 Hz). MALDI-TOF-MS, 979.0 (979.1 calc for M+H).

19. ImPyPy-G-PyPyPy-β-Dp-NH₂:

Polyamide was prepared by manual solid phase methods as a white powderupon cleavage of 240 mg resin with N-methyl-bis(aminopropyl)amine (2 ml,55° C.) (25 mg, 55% recovery). HPLC r.t. 22.0; ¹H NMR (DMSO-d₆) δ 10.53(s, 1 H), 10.00 (s, 1 H), 9.98 (s, 1 H), 9.93 (s, 1 H), 9.92 (s, 1 H),9.7 (br s, 1 H), 8.31 (t, 1 H, J=5.7 Hz), 8.12 (t, 1 H, J=5.5 Hz), 8.04(t, 1 H, J=5.6 Hz), 7.9 (br s, 3 H), 7.41 (s, 1 H), 7.29 (d, 1 H, J=1.7Hz), 7.23 (d, 1 H, J=1.5 Hz), 7.22 (d, 1 H, J=1.4 Hz), 7.16 (m, 3 H),7.07 (d, 1 H, J=1.2 Hz), 7.03 (d, 1 H, J=1.3 Hz), 6.94 (d, 1 H, J=1.6Hz), 6.93 (d, 1 H, J=1.5 Hz), 6.86 (d, 1 H, J=1.4 Hz), 3.98 (s, 3 H),3.88 (d, 2 H, J=5.6 Hz), 3.83 (s, 3 H), 3.82 (m, 6 H), 3.80 (s, 3 H),3.78 (s, 3 H), 3.37 (q, 2 H, J=6.4 Hz), 3.11 (m, 6 H), 2.86 (q, 2 H,J=6.1 Hz), 2.70 (d, 3 H, J=4.6 Hz),2.32 (t, 2 H, J=7.2 Hz), 1.87(quintet, 2 H, J=7.4 Hz), 1.75 (quintet, 2 H, J=6.0 Hz), MALDI-TOF-MS,993.3 (993.1 calc for M+H).

20. ImPyPy-β-PyPyPy-G-Dp-NH₂:

Polyamide was prepared by automated solid phase methods as a whitepowder upon cleavage of 240 mg resin with N-methyl-bis(aminopropyl)amine(2 ml, 55° C.) (23.0 mg, 53% recovery). HPLC, r.t. 20.6; ¹H NMR(DMSO-d₆) δ 10.45 (s, 1 H), 9.95 (s, 1 H), 9.92 (m, 3 H), 9.6 (br s, 1H), 8.27 (t, 1 H, J=4.7 Hz), 8.11 (m, 2 H), 7.9 (s, 3 H), 7.38 (s, 1 H),7.26 (d, 1 H, J=1.7 Hz), 7.21 (m, 2 H), 7.17 (m, 2 H), 7.13 (d, 1 H,J=1.8 Hz), 7.05 (m, 2 H), 6.93 (d, 1 H, J=1.6 Hz), 6.88 (d, 1 H, J=1.6Hz), 6.83 (d, 1 H, J=1.7 Hz), 3.97 (s, 3 H), 3.82 (s, 9 H), 3.81 (s, 3H), 3.79 (s, 3 H), 3.73 (m, 2 H), 3.44 (q, 2 H, J=5.5 Hz), 3.2 (m, 6 H),2.85 (q, 2 H, J=5.8 Hz), 2.73 (d, 3 H, J=4.5 Hz), 1.89 (quintet, 2 H,J=6.4 Hz), 1.77 (quintet, 2 H, J=6.9 Hz), MALDI-TOF-MS, 992.9 (993.1calc for M+H).

21. ImPyPy-γ-ImPyPy-β-PyPyPy-G-Dp-NH₂:

The polyamide was prepared by machine-assisted solid phase methods as awhite powder. (29 mg 59% recovery). HPLC r.t. 21.5, ¹H NMR (DMSO-d₆); δ10.50 (s, 1 H), 10.27 (s, 1 H), 9.96 (s, 1 H), 9.93 (m, 5 H), 9.2 (br s,1 H), 8.27 (t, 1 H), J=5.1 Hz), 8.03 (m, 3 H), 7.90 (s, 3 H), 7.45 (s, 1H), 7.40 (s, 1 H), 7.27 (d, 1 H, J=1.3 Hz), 7.25 (d, 1 H, J=1.4 Hz),7.22 (m, 2 H), 7.18 (m, 2 H), 7.17 (d, 1 H, J=1.4 Hz), 7.14 (d, 1 H,J=1.3 Hz), 7.11 (m, 2 H), 7.06 (d, 1 H, J=1.5 Hz), 6.94 (d, 1 H, J=1.3Hz), 6.88 (m, 2 H), 6.84 (d, 1 H, J=1.4 Hz), 3.97 (s, 3 H), 3.93 (s, 3H), 3.83 (m, 9 H), 3.80 (m, 6 H), 3.76 (m, 6 H), 3.72 (d, 2 H, J=5.2Hz), 3.43 (q, 2 H, J=5.0 Hz), 3.17 (m, 6 H), 3.11 (q, 2 H, J=5.3 Hz),2.85 (q, 2 H, J=5.2 Hz), 2.73 (d, 3 H, J=3.9 Hz), 2.51 (t, 2 H, J=6.5Hz), 2.35 (t, 2 H, J=6.7 Hz), 1.92 (quintet, 2 H, J=6.8 Hz), 1.78 (m, 4H). MALDI-TOF MS 1445.6 (1445.6 calc for M+H).

22. ImImImPy-γ-PyPyPyPy-β-Dp-NH₂ :

A sample of machine-synthesized resin (350 mg, 0.16 mmol/gram) wasplaced in a 20 mL glass scintillation vial, and treated with 2 mL3,3′-diamino-N-methyldipropylamine at 55° C. for 18 hours. The resin wasremoved by filtration through a disposable propylene filter, and theresulting solution dissolved with water to a total volume of 8 mL, andpurified directly by preparatory reversed phase HPLC to provideImImImPy-γ-PyPyPyPy-β-Dp-NH₂ (31 mg, 40% recovery) as a white powder. ¹HNMR (DMSO-d₆) δ 10.37 (s, 1 H), 10.16 (s, 1 H), 9.95 (s, 1 H), 9.93 (s,1 H), 9.91 (s, 1 H), 9.86 (s, 1 H); 9.49 (br s, 1 H), 9.47 (s, 1 H),8.12 (m, 3 H), 8.0 (br s, 3 H), 7.65 (s, 1 H), 7.57 (s, 1 H), 7.46 (s, 1H), 7.20 (m, 3 H), 7.16 (m, 2 H), 7.09 (d, 1 H, J=1.5 Hz), 7.05 (m, 2H), 7.00 (d, 1 H, J=1.6 Hz), 6.88 (m, 2 H), 4.01 (s, 3 H), 3.99 (s, 3H), 3.98 (s, 3 H), 3.83 (s, 3 H), 3.82 (s, 3 H), 3.81 (s, 3 H), 3.79 (s,3 H), 3.78 (s, 3 H), 3.36 (q, 2 H, J=5.3 Hz), 3.21-3.05 (m, 8 H), 2.85(q, 2 H, J=4.9 Hz), 2.71 (d, 3 H, J=4.4 Hz), 2.34 (t, 2 H, J=5.9 Hz),2.26 (t, 2 H, J=5.9 Hz), 1.85 (quintet, J=5.7 Hz), 1.72 (m, 4 H).MALDI-TOF-MS, 1267.1 (1267.4 calc. for M+H).

23. ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp-NH₂:

A sample of ImPyPyPyPy-γ-ImPyPyPyPy-β-resin prepared by machine-assistedsolid phase synthesis (240 mg, 0.16 mmol/gram) was placed in a 20 mLglass scintillation vial, and treated with3,3-diamino-N-methyldipropylamine (2 mL) at 55° C. for 18 hours. Resinwas removed by filtration, and the filtrate diluted to a total volume of8 mL with 0.1% (wt/v) aqueous TFA. The resulting crude polyamide/aminesolution was purified directly by reversed phase HPLC to provide thetrifluoroacetate salt of ImPyPyPyPy-γ-ImPyPyPyPy-β-NH₂ (31 mg, 40%recovery) as a white powder. UV λ_(max) 241, 316 (ε) 83300 (calculatedbased on ε=8,333/ring⁵); ¹H NMR (DMSO-d₆) δ 10.53 (s, 1 H), 10.28 (s, 1H), 10.03 (s, 1 H), 10.00 (s, 1 H), 9.96 (m, 2 H), 9.92 (m, 2 H), 9.6(br s, 1 H), 8.07 (m, 4 H), 7.89 (s, 3 H), 7.45 (s, 1 H), 7.41 (s, 1 H),7.27 (d, 2 H, J=8.5 Hz), 7.23 (m, 4 H), 7.16 (m, 4 H), 7.06 (m, 4 H),6.87 (m, 2 H), 3.98, (s, 3 H), 3.94 (s, 3 H), 3.84, (m, 6 H), 3.79 (s, 3H), 3.35 (q, 2 H, J=5.7 Hz), 3.16 (m, 8 H), 2.85 (q, 2 H, J=5.6 Hz),2.72 (d, 2 H, J=4.2 Hz), 2.34 (m, 2 H), 1.91 (m, 4 H), 1.78 (m, 4 H).MALDI-TOF MS, 1510.4 (1510.7 calc. for M+H).

24. ImImPyPyPy-γ-ImPyPyPyPy-β-Dp-NH₂:

The polyamide was prepared as a white powder as described forImPyPyPyPy-γ-ImPyPyPyPy-β-NH₂. ¹H NMR (DMSO-d₆) δ 10.39 (s, 1 H), 10.28(s, 1 H), 10.03 (s, 1 H), 10.00 (s, 1 H), 9.92 (m, 2 H), 9.82 (s, 1 H),9.66 (br s, 1 H), 8.11 (m, 4 H), 7.89 (s, 3 H), 7.57 (s, 1 H), 7.46 (d,2 H, J=2.4 Hz), 7.27 (dd, 2 H, J=1.0 Hz) 7.32 (m, 4 H), 7.16 (m, 4 H),7.08 (m, 4 H), 6.88 (m, 1 H), 4.00 (s, 3 H), 3.94 (s, 3 H), 3.78 (s, 3H), 3.19 (q, 2 H, J=5.1 Hz), 3.05 (m, 8 H), 2.86 (q, 2 H, J=4.8 Hz),2.72 (d, 2 H, J=4.4 Hz), 2.34 (m, 4 H), 1.90 (m, 4 H), 1.78 (m, 4 H).MALDI-TOF-MS, 1510.4 (1511.7 calc. for M+H).

25. ImImIm-γ-PyPyPy-β-Dp-EDTA:

EDTA-dianhydride (50 mg) was dissolved in 1 mL DMSO/NMP solution and 1mL DIEA by heating at 55° C. for 5 min. The dianhydride solution wasadded to ImImIm-γ-PyPyPy-β-Dp-NH₂ (8.0 mg, 7 μmol) dissolved in 750 μLDMSO. The mixture was heated at 55° C. for 25 minutes, and treated with3 mL 0.1M NaOH, and heated at 55° C. for 10 minutes. 0.1% TFA was addedto adjust the total volume to 8 mL and the solution purified directly bypreparatory HPLC chromatography to provide ImImIm-γ-PyPyPy-β-Dp-EDTA asa white powder. (3.3 mg, 30% recovery) ¹H NMR (DMSO-d₆) d 10.14 (s, 1H), 9.90 (s, 1 H), 9.89 (s, 1 H), 9.85 (s, 1 H), 9.58 (s, 1 H), 9.3 (brs, 1 H), 8.40 (m, 2 H), 8.02 (m, 2 H), 7.65 (s, 1 H), 7.51 (s, 1 H),7.45 (s, 1 H), 7.20 (d, 1 H, J=1.5 Hz), 7.15 (m, 2 H), 7.08 (d, 1 H,J=1.1 Hz), 7.04 (d, 1 H, J=1.5 Hz), 6.86 (m, 2 H), 4.00 (s, 3 H), 3.98(s, 3 H), 3.94 (s, 3 H), 3.87 (m, 4 H), 3.82 (s, 3 H), 3.81 (s, 3 H),3.78 (s, 3 H), 3.72 (m, 4 H), 3.4-3.0 (m, 16 H), 2.71 (d, 3 H, J=4.2Hz), 2.33 (t, 2 H, J=5.1 Hz), 2.25 (t, 2 H, J=5.9 Hz), 1.75 (m, 6 H).MALDI-TOF-MS, 1298.4 (1298.3 calc. for M+H). The polyamide was loadedwith Fe(II) by standard methods.

26. ImPyPy-γ-ImPyPy-β-Dp:

The polyamide was prepared by machine-assisted solid phase methods as awhite powder. (17 mg, 56% recovery). HPLC r.t. 26.1, UV λ_(max) (ε), 234(39,300), 312 (53,200) nm; ¹H NMR (DMSO-d₆); d 10.53 (s, 1 H), 10.27 (s,1 H), 10.04 (s, 1 H), 9.96 (s, 1 H), 9.94 (s, 1 H), 9.2 (br s, 1 H),8.08 (m, 3 H), 7.49 (s, 2 H), 7.44 (s, 1 H), 7.31 (d, 1 H, J=1.0 Hz),7.23 (d, 1 H, J=1.1 Hz), 7.19 (m, 3 H), 7.10 (s, 1 H), 6.92 (d, 1 H,J=1.1 Hz), 6.90 (d, 1 H, J=1.1 Hz). 4.01 (s, 3 H), 3.97 (s, 3 H), 3.86(m, 6 H), 3.82 (m, 6 H), 3.41 (q, 2 H, J=6.0 Hz), 3.22 (q, 2 H, J=5.9Hz), 3.13 (q, 2 H, J=5.9 Hz), 3.0 (q, 2 H, J=5.6 Hz), 2.76 (d, 6 H,J=4.8 Hz), 2.37 (m, 4 H), 1.78 (m, 4 H); MALDI-TOF MS 979.3 (979.1 calc.for M+H).

27. ImPyPy-G-PyPyPy-G-Dp:

Polyamide was prepared by manual solid phase methods and obtained as awhite powder upon cleavage of 240 mg resin. (initial subsitution of 0.2mmol Boc-Glycine/gram) with dimethylaminopropylamine (11.9 mg, 29%recovery). HPLC, r.t. 26.9 min.; UV λ_(max) (ε), 246 (41,000), 312(48,400) nm; ¹HNMR (DMSO-d₆) δ 10.49 (s, 1 H), 9.98 (s, 1 H), 9.95 (s, 1H), 9.92 (s, 1 H), 9.89 (s, 1 H), 9.2 (br s, 1 H), 8.30 (m, 2 H), 8.06(t, 1 H, J=5.8 Hz), 7.40 (s, 1 H), 7.24, (d, 1 H, J=1.7 Hz), 7.23 (m, 3H), 7.17 (m, 2 H), 7.06 (m, 2 H), 6.94 (m, 3 H), 3.99 (s, 3 H), 3.89 (d,2 H, J=5.8 Hz), 3.84 (s, 3 H), 3.84 (s, 3 H), 3.83 (s, 3 H), 3.81 (s, 3H), 3.80 (s, 3 H), 3.72 (d, 2 H, J=4.3 Hz), 3.13 (q, 2 H, J=5.7 Hz),3.01 (q, 2 H, J=5.2 Hz), 2.76 (d, 6 H, J=4.3 Hz), 1.77 (quintet, 2 H,J=7.4 Hz); MALDI-TOF MS 935.7 (M+H 936.0 calc for C₄₄H₅₅N₁₆O₈); FABMSm/e 935.433 (M+H 935.439 calcd. for C₄₄H₅₅N₁₆O₈).

28. ImPyPy-G-PyPyPy-β-Dp:

Polyamide was prepared by manual solid phase methods as a white powderupon cleavage of 180 mg resin (initial subsitution of 0.2 mmolBoc-β-alanine/gram) with dimethylaminopropylamine (2 mL, 55° C.). (12.3mg, 38% recovery after HPLC purification). HPLC, r.t. 25.5, UV λ_(max)(ε), 246 (39,500), 312 (52,000) nm; ¹H NMR (DMSO-d₆); 10.46 (s, 1 H),9.96 (s, 1 H), 9.90 (s, 1 H), 9.88 (m, 2 H), 9.21 (br s, 1 H), 8.27 (t,1 H, J=4.2 Hz), 8.06 (m, 2 H), 7.39 (s, 1 H), 7.28 (d, 1 H, J=1.6 Hz),7.23 (d, 1 H, J=1.7 Hz), 7.20 (d, 1 H, J=1.5 Hz), 7.15 (m, 3 H), 7.04(m, 2 H), 7.03 (d, 1 H, J=1.6 Hz), 6.94 (d, 1 H, J=1.7 Hz), 6.92 (d, 1H, J=1.4 Hz), 3.98 (s, 3 H), 3.88 (d, 2 H, J=5.6 Hz), 3.83 (s, 3 H),3.82 (m, 6 H), 3.79 (s, 3 H), 3.78 (s, 3 H), 3.36 (q, 2 H, J=5.3 Hz),3.09 (q, 2 H, J=6.0 Hz), 2.75 (q, 2 H, J=5.2 Hz), 2.72 (d, 6 H, J=4.8Hz), 2.30 (t, 2 H, J=6.3 Hz), 1.72 (quintet, 2 H, J=5.7 Hz) MALDI-TOF MS950.1 (950.0 calc for M+H); FABMS m/e 949.462 (M+H 949.455 calc. forC₄₅H₅₇N₁₆O₈).

29. ImPyPy-γ-PyPyPy-G-Dp:

Polyamide was prepared by automated solid phase methods as a whitepowder upon cleavage of 180 mg resin (initial subsitution of 0.2 mmolBoc-Glycine/gram) with dimethylaminopropylamine (2 ml, 55° C.) (17.2 mg,57% recovery after HPLC purification). HPLC, r.t. 26.5; UV λ_(max) (ε),246 (46,500), 312 (54,800) nm; ¹H NMR (DMSO-d₆) δ 10.54 (s, 1 H), 9.92(s, 1 H), 9.90 (m, 3 H), 9.23 (br s, 1 H), 8.27 (t, 1 H, J=5.5 Hz), 8.06(t, 1 H, J=6.3 Hz), 8.03 (t, 1 H, J=6.2 Hz), 7.39 (s, 1 H), 7.26 (d, 1H, J=1.7 Hz), 7.20 (m, 2 H), 7.17 (m, 3 H), 7.13 (m, 2 H), 7.04 (d, 1 H,J=1.5 Hz), 6.87 (d, 1 H, J=1.8 Hz), 6.83 (d, 1 H, J=1.8 Hz), 3.97 (s, 3H), 3.82 (m, 15 H), 3.78 (d, 2 H, J=3.4 Hz), 3.27 (m, 4 H), 3.15 (m, 2H), 3.79 (m, 2 H), 2.76 (d, 6 H, J=4.9 Hz), 1.78 (quintet, 2 H, J=6.6Hz) MALDI-TOF MS 950.2 (950.0 calc. for M+H); FABMS m/e 949.458 (M+H949.455 calc. for C₄₅H₅₇N₁₆O₈).

30. ImPyPy-β-PyPyPy-β-Dp:

Polyamide was prepared by automated solid phase methods as a whitepowder upon cleavage of 240 mg resin (initial subsitution of 0.2 mmolBoc-β-alanine/gram) with dimethylaminopropylamine (2 ml, 55° C.). (19.0mg, 43% recovery after HPLC purification). HPLC, r.t. 26.8; UV λ_(max)(ε), 246 (42,100), 312 (53,900) nm; ¹H NMR (DMSO-d₆) δ 10.56 (s, 1 H),9.90 (s, 1 H), 9.89 (m, 2 H), 9.87 (s, 1 H), 9.21 (br s, 1 H), 8.24 (t,1 H, J=5.2 Hz), 8.04 (t, 1 H, J=6.1 Hz), 8.01 (t, 1 H, J=6.0 Hz), 7.35(s, 1 H), 7.26 (d, 1 H, J=1.6 Hz), 7.23 (m, 3 H), 7.16 (m, 3 H), 7.12(m, 1 H), 7.02 (d, 1 H, J=1.5 Hz), 6.85 (d, 1 H, J=1.9 Hz), 6.80 (d, 1H, J=1.8 Hz), 3.96 (s, 3 H), 3.79 (s, 3 H), 3.78 (s, 3 H), 3.36 (q, 2 H,J=5.3 Hz), 3.09 (q, 2 H, J=6.0 Hz), 2.75 (q, 2 H, J=5.0 Hz), 2.72 (d, 6H, J=4.7 Hz), 2.30 (t, 2 H, J=6.1 Hz), 1.72 (quintet, 2 H, J=5.5 Hz);MALDI-TOF MS 964.2 (964.1 calc. for M+H)

31. ImPyPy-Py-PyPyPy-G-Dp:

Polyamide was prepared by manual solid phase methods. Recovery is basedon cleavage of 180 mg resin (initial subsitution of 0.2 mmolBoc-Glycine/gram) with dimethylaminopropylamine (2 ml, 55° C.). (8 mg,24% recovery after HPLC purification). A small quantity of the failureheptamide AcPyPyPyPyPyPy-Dp was found in the initial preparation and wasremoved by a second preparatory HPLC purification to afford pureImPyPy-Py-PyPyPy-G-Dp as a white powder (1.2 mg). HPLC, r.t. 28.5, UVλ_(max) (ε), 246 (34,600), 312 (55,300); ¹H NMR (DMSO-d₆) δ 10.55 (s, 1H), 10.02 (s, 1 H), 10.00 (m, 4 H), 9.3 (br s, 1 H), 8.32 (t, 1 H, J=6.2Hz), 8.06 (t, 1 H, J=5.9 Hz), 7.44 (s, 1 H), 7.31 (d, 1 H, J=1.7 Hz),7.26 (m, 5 H), 7.19 (d, 1 H, J=1.8 Hz), 7.10 (m, 5 H), 6.97 (d, 1 H,J=1.7 Hz), 4.01 (s, 3 H), 3.87 (m, 15 H), 3.82 (s, 3 H), 3.73 (d, 2 H,J=5.5 Hz), 3.16 (q, 2 H, J=6.2 Hz), 3.03 (q, 2 H, J=5.2 Hz), 2.74 (d, 6H, J=4.9 Hz), 1.77 (quintet, 2 H, J=6.7 Hz); MALDI-TOF MS 1000.5; FABMSm/e 1001.471 (M+H 1001.473 calcd. for C₄₈H₅₉N₁₇O₈).

32. ImPyPy-Py-PyPyPy-β-Dp:

Polyamide was prepared by machine assisted solid phase synthesis toafford a white powder upon cleavage of 800 mg resin (initial subsitutionof 0.2 mmol Boc-β-alanine/gram) with dimethylaminopropylamine (2 ml, 55°C.). (56 mg, 36% recovery after HPLC purification) (ε) 246 (34,800), 308(57,000); HPLC r.t. 27.9 min.; ¹H NMR (DMSO-d₆) δ 10.47 (s, 1 H), 9.95(m, 4 H), 9.89 (s, 1 H), 9.2 (br s, 1 H), 8.03 (m, 2 H), 7.39 (s, 1 H),7.27 (d, 1 H, J=1.3 Hz), 7.22 (m, 4 H), 7.15 (m, 2 H), 7.07 (m, 4 H),7.03 (d, 1 H, J=1.4 Hz), 6.86 (d, 1 H, J=1.0 Hz), 3.97 (s, 3 H), 3.84(m, 12 H), 3.82 (s, 3 H), 3.77 (s, 3 H), (β-ala quartet covered bywater.), 3.11 (q, 2 H, J=5.1 Hz), 3.08 (q, 2 H, J=6.0 Hz), 2.72 (d, 6 H,J=4.8 Hz), 2.34 (t, 2 H, J=4.4 Hz), 1.7 (m, 2 H); MALDI-TOF-MS, 1014.7(1015.1 calc for M+H).

33. ImPyPy-G-PyPyPy-G-Dp:

Polyamide was prepared by manual solid phase methods as a white powderupon cleavage of 240 mg resin with N-methyl-bis(aminopropyl)amine (2 ml,55° C.) (19.0 mg, 44% recovery after HPLC purification). ¹H NMR(DMSO-d₆) δ 10.49 (s, 1 H), 9.97 (s, 1 H), 9.93 (s, 1 H), 9.91 (s, 1 H),9.89 (s, 1 H), 9.7 (br s, 1 H), 8.27 (m, 2 H), 8.04 (t, 1 H, J=5.1 Hz),7.88 (br s, 3 H), 7.39 (s, 1 H), 7.27 (d, 1 H, J=1.6 Hz), 7.21 (m, 3 H),7.15 (m, 2 H), 7.05 (m, 2 H), 6.93 (m, 3 H), 3.97 (s, 3 H), 3.96 (m, 6H), 3.92 (m, 9 H), 3.72 (m, 4 H), 3.14 (m, 6 H), 3.05 (q, 2 H, J=5.4Hz), 2.73 (d, 3 H, J=3.3 Hz), 1.88 (quintet, 2 H, J=4.6 Hz), 1.75(quintet, 2 H, J=6.3 Hz). MALDI-TOF-MS, 979.0 (979.1 calc for M+H).

34. ImPyPy-G-PyPyPy-β-Bp:

Polyamide was prepared by manual solid phase methods as a white powderupon cleavage of 240 mg resin with N-methyl-bis(aminopropyl)amine (2 ml,55° C.) (25 mg, 55% recovery). HPLC, r.t. 22.0; ¹H NMR (DMSO-d₆) δ 10.53(s, 1 H), 10.00 (s, 1 H), 9.98 (s, 1 H), 9.93 (s, 1 H), 9.92 (s, 1 H),9.7 (br s, 1 H), 8.31 (t, 1 H, J=5.7 Hz), 8.12 (t, 1 H, J=5.5 Hz), 8.04(t, 1 H, J=5.6 Hz), 7.9 (br s, 3 H), 7.41 (s, 1 H), 7.29 (d, 1 H, J=1.7Hz), 7.23 (d, 1 H, J=1.5 Hz), 7.22 (d, 1 H, J=1.4 Hz), 7.16 (m, 3 H),7.07 (d, 1 H, J=1.2 Hz), 7.03 (d, 1 H, J=1.3 Hz), 6.94 (d, 1 H, J=1.6Hz), 6.93 (d, 1 H, J=1.5 Hz), 6.86 (d, 1 H, J=1.4 Hz), 3.98 (s, 3 H),3.88 (d, 2 H, J=5.6 Hz), 3.83 (s, 3 H), 3.82 (m, 6 H), 3.80 (s, 3 H),3.78 (s, 3 H), 3.37 (q, 2 H, J=6.4 Hz), 3.11 (m, 6 H), 2.86 (q, 2 H,J=6.1 Hz), 2.70 (d, 3 H, J=4.6 Hz), 2.32 (t, 2 H, J=7.2 Hz), 1.87(quintet, 2 H, J=7.4 Hz), 1.75 (quintet, 2 H, J=6.0 Hz), MALDI-TOF-MS,993.3 (993.1 calc for M+H).

35. ImPyPy-β-PyPyPy-G-Dp:

Polyamide was prepared by automated solid phase methods as a whitepowder upon cleavage of 240 mg resin with N-methyl-bis(aminopropyl)amine(2 ml, 55° C.) (23.0 mg, 53% recovery). HPLC, r.t. 20.6; ¹H NMR(DMSO-d₆) δ 10.45 (s, 1 H), 9.95 (s, 1 H), 9.92 (m, 3 H), 9.6 (br s, 1H), 8.27 (t, 1 H, J=4.7 Hz), 8.11 (m, 2 H), 7.9 (s, 3 H), 7.38 (s, 1 H),7.26 (d, 1 H, J=1.7 Hz), 7.21 (m, 2 H), 7.17 (m, 2 H), 7.13 (d, 1 H,J=1.8 Hz), 7.05 (m, 2 H), 6.93 (d, 1 H, J=1.6 Hz), 6.88 (d, 1 H, J=1.6Hz), 6.83 (d, 1 H, J=1.7 Hz), 3.97 (s, 3 H), 3.82 (s, 9 H), 3.81 (s, 3H), 3.79 (s, 3 H), 3.73 (m, 2 H), 3.44 (q, 2 H, J=5.5 Hz), 3.2 (m, 6 H),2.85 (q, 2 H, J=5.8 Hz), 2.73 (d, 3 H, J=4.5 Hz), 1.89 (quintet, 2 H,J=6.4 Hz), 1.77 (quintet, 2 H, J=6.9 Hz) MALDI-TOF-MS, 992.9 (993.1 calcfor M+H).

36. ImPyPy-G-PyPyPy-G-Dp-EDTA:

EDTA-dianhydride (50 mg) was dissolved in 1 mL DMSO/NMP solution and 1mL DIEA by heating at 55° C. for 5 min. The dianhydride solution wasadded to ImPyPy-G-PyPyPy-G-Bp (12.0 mg, 11 μmol) dissolved in 750 μLDMSO. The mixture was heated at 55° C. for 25 minutes, and treated with3 mL 0.1M NaOH, and heated at 55° C. for 10 minutes. 0.1% TFA was addedto adjust the total volume to 8 mL and the solution purified directly bypreparatory HPLC chromatography to provide ImPyPy-G-PyPyPy-G-Bp-EDTA asa white powder. (4.7 mg, 31% recovery after HPLC purification); HPLC,r.t. 28.8; ¹H NMR (DMSO-d₆) δ 10.49 (s, 1 H), 9.97 (s, 1 H), 9.91 (s, 1H), 9.89 (m, 2 H), 9.4 (br s, 1 H), 8.42 (t, 1 H, J=5.0 Hz), 8.31 (t, 1H, J=5.5 Hz), 8.00 (m, 2 H), 7.38 (s, 1 H), 7.26 (d, 1 H, J=1.5 Hz),7.22 (d, 1 H, J=1.4 Hz), 7.20 (d, 1 H, J=1.4 Hz), 7.14 (m, 3 H), 7.03(m, 2 H), 6.92 (d, 1 H, J=1.5 Hz), 3.95 (s, 3 H), 3.85 (m, 4 H), 3.84(s, 3 H), 3.80 (m, 6 H), 3.78 (s, 3 H), 3.76 (s, 3 H), 3.69 (m, 6 H),3.55 (q, 2 H, J=5.7 Hz), 3.3-3.0 (m, 12 H), 2.69 (d, 3 H, J=3.9 Hz),2.31 (t, 2 H, J=6.8 Hz), 1.73 (m, 4 H); MALDI-TOF-MS, 1254.8 (1254.3calc for M+H).

37. ImPyPy-G-PyPyPy-β-Bp-EDTA:

Polyamide was prepared from ImPyPy-G-PyPyPy-β-Bp (20 mg) as describedfor ImPyPy-G-PyPyPy-G-Bp-EDTA. (13.0 mg, 55% recovery after HPLCpurification). HPLC, r.t. 27.3; ¹H NMR (DMSO-d₆) δ 10.49 (s, 1 H), 9.97(s, 1 H), 9.91 (s, 1 H), 9.89 (m, 2 H), 9.4 (br s, 1 H), 8.42 (t, 1 H,J=5.0 Hz), 8.31 (t, 1 H, J=5.5 Hz), 8.00 (m, 2 H), 7.38 (s, 1 H), 7.26(d, 1 H, J=1.5 Hz), 7.22 (d, 1 H, J=1.4 Hz), 7.20 (d, 1 H, J=1.4 Hz),7.14 (m, 3 H), 7.03 (m, 2 H), 6.92 (d, 1 H, J=1.5 Hz), 3.95 (s, 3 H),3.85 (m, 4 H), 3.84 (s, 3 H), 3.80 (m, 6 H), 3.78 (s, 3 H), 3.76 (s, 3H), 3.69 (m, 6 H), 3.55 (q, 2 H, J=5.7 Hz), 3.3-3.0 (m, 12 H), 2.69 (d,3 H, J=3.9 Hz), 2.31 (t, 2 H, J=6.8 Hz), 1.73 (m, 4 H); MALDI-TOF-MS,1268.5 (1268.3 calc for M+H).

38. ImPyPy-β-PyPyPy-G-Bp-EDTA:

Polyamide was prepared from ImPyPy-β-PyPyPy-G-Bp (12 mg) as describedfor ImPyPy-G-PyPyPy-G-Bp-EDTA. (6 mg, 42% recovery after HPLCpurification). HPLC, r.t. 28.0; ¹H NMR (DMSO-d₆) δ 10.46 (s, 1 H), 9.95(s, 1 H), 9.93 (m, 3 H), 9.9 (br s, 1 H), 8.43 (t, 1 H, J=5.1 Hz), 8.28(t, 1 H, J=5.3 Hz), 8.03 (m, 2 H), 7.38 (s, 1 H), 7.26 (m, 2 H), 7.21(d, 1 H, J=1.6 Hz), 7.17 (d, 1 H, J=1.8 Hz), 7.12 (d, 1 H, J=1.8 Hz),7.10 (s, 1 H), 7.04 (d, 1 H, J=1.6 Hz), 6.93 (m, 2 H), 6.88 (d, 1 H,J=1.6 Hz), 6.84 (d, 1 H, J=1.4 Hz), 3.97 (s, 3 H), 3.87 (m, 4 H), 3.82(m, 9 H), 3.79 (s, 3 H), 3.78 (s, 3 H), 3.68 (m, 6 H), 3.3-2.9 (m, 16H), 2.71 (d, 3 H, J=4.1 Hz), 1.78 (m, 4 H); MALDI-TOF-MS, 1268.9 (1268.3calc for M+H).

39. ImPyPy-γ-ImPyPy-β-PyPyPy-G-Dp:

The polyamide was prepared by machine-assisted solid phase methods as awhite powder. (12 mg 19% recovery). HPLC r.t. 29.5, UV λ_(max) (ε), 238(53,900), 312 (71,100) nm; ¹H NMR (DMSO-d₆); d 10.46 (s, 1 H), 10.24 (s,1 H), 9.96 (s, 1 H), 9.90 (m, 5 H), 9.2 (br s, 1 H), 8.25 (m, 1 H), 8.00(m, 3 H), 7.44 (s, 1 H), 7.39 (s, 1 H), 7.26 (d, 1 H, J=1.3 Hz), 7.24(d, 1 H, J=1.5 Hz), 7.20 (m, 2 H), 7.16 (m, 2 H), 7.13 (m, 2 H), 7.11(d, 1 H, J=1.4 Hz), 7.05 (d, 1 H, J=1.4 Hz), 7.03 (d, 1 H, J=1.5 Hz),6.93 (d, 1 H, J=1.3 Hz), 6.87 (m, 2 H), 6.84 (d, 1 H, J=1.5 Hz), 3.97(s, 3 H), 3.92 (s, 3 H), 3.82 (m, 9 H), 3.79 (m, 6 H), 3.76 (m, 6 H),3.73 (m, 2 H), 3.44 (q, 2 H, J=5.0 Hz), 3.17 (m, 4 H), 3.03 (m, 2 H),2.74 (d, 6 H, J=4.8 Hz), 2.50 (m, 2 H), 2.33 (t, 2 H, J=6.7 Hz), 1.77(m, 4 H). MALDI-TOF MS 1402.2 (1402.5 calc for M+H).

40. ImPyPy-γ-ImPyPy-β-PyPyPy-G-Dp-NH₃

The polyamide was prepared by machine-assisted solid phase methods as awhite powder. (29 mg 59% recovery). HPLC r.t. 21.5, ¹H NMR (DMSO-d₆); δ10.50 (s, 1 H), 10.27 (s, 1 H), 9.96 (s, 1 H), 9.93 (m, 5 H), 9.2 (br s,1 H), 8.27 (t, 1 H, J=5.1 Hz), 8.03 (m, 3 H), 7.90 (s, 3 H), 7.45 (s, 1H), 7.40 (s, 1 H), 7.27 (d, 1 H, J=1.3 Hz), 7.25 (d, 1 H, J=1.4 Hz),7.22 (m, 2 H), 7.18 (m, 2 H), 7.17 (d, 1 H, J=1.4 Hz), 7.14 (d, 1 H,J=1.3 Hz), 7.11 (m, 2 H), 7.06 (d, 1 H, J=1.5 Hz), 6.94 (d, 1 H, J=1.3Hz), 6.88 (m, 2 H), 6.84 (d, 1 H, J=1.4 Hz), 3.97 (s, 3 H), 3.93 (s, 3H), 3.83 (m, 9 H), 3.80 (m, 6 H), 3.76 (m, 6 H), 3.72 (d, 2 H, J=5.2Hz), 3.43 (q, 2 H, J=5.0 Hz), 3.17 (m, 6 H), 3.11 (q, 2 H, J=5.3 Hz),2.85 (q, 2 H, J=5.2 Hz), 2.73 (d, 3 H, J=3.9 Hz), 2.51 (t, 2 H, J=6.5Hz), 2.35 (t, 2 H, J=6.7 Hz), 1.92 (quintet, 2 H, J=6.8 Hz), 1.78 (m, 4H). MALDI-TOF MS 1445.6 (1445.6 calc for M+H).

41. ImPyPy-γ-ImPyPy-β-PyPyPy-G-Dp-EDTA:

EDTA-dianhydride (50 mg) was dissolved in 1 mL DMSO/NMP solution and 1mL DIEA by heating at 55° C. for 5 min. The dianhydride solution wasadded to ImPyPy-γ-ImPyPy-β-PyPyPy-G-Dp-NH₂ (9.0 mg, 5 μmol) dissolved in750 μL DMSO. The mixture was heated at 55° C. for 25 minutes, andtreated with 3 mL 0.1M NaOH, and heated at 55° C. for 10 minutes. 0.1%TFA was added to adjust the total volume to 8 mL and the solutionpurified directly by reversed-phase HPLC to provideImPyPy-γ-ImPyPy-β-PyPyPy-G-Dp-EDTA as a white powder. (3 mg, 30%recovery after HPLC purification); MALDI-TOF MS 1720.1 (1719.8 calc forM+H).

42. Ac-PyImPy-γ-ImPyPy-β-PyPyPy-β-Dp:

The polyamide was prepared by machine assisted solid phase methods (29)as a white powder (5 mg, 20% recovery). UV (H2O) λ_(max) 242 nm, 310 nm(ε=75,000, calculated based on e=8333/ring (30)); ¹H NMR (DMSO-d₆): δ10.27 (m, 2 H); 10.02 (s, 1 H); 9.99 (s, 1 H), 9.92 (m, 3 H), 9.90 (s, 1H), 9.85 (s, 1 H), 8.6 (br s, 1 H), 8.08 (m, 4 H), 7.52 (s, 1 H); 7.43(s, 1 H); 7.24 (m, 2 H), 7.22 (d, 1 H, J=1.7 Hz), 7.20 (d, 1 H, J=1.6Hz), 7.19 (d, 1 H, J=1.6 Hz), 7.14 (d, 1 H, J=1.5 Hz), 7.11 (d, 1 H,J=1.6 Hz), 7.07 (m, 2 H), 7.02 (d, 1 H, J=1.4 Hz), 6.96 (d, 1 H, J=1.7Hz), 6.90 (m, 2 H), 6.88 (d, 1 H, J=1.8 Hz), 6.83 (d, 1 H, J=1.6 Hz),3.94 (s, 3 H), 3.92 (s, 3 H), 3.81 (m, 12 H), 3.79 (s, 3 H), 3.78 (s, 3H), 3.78 (s, 3 H), 3.52 (m, 4 H), 3.33 (m, 6 H), 2.95 (m, 2 H), 2.71 (d,6 H, J=4.7 Hz), 2.32 (m, 4 H), 1.94 (s, 3 H), 1.73 (m, 4 H). MALDI-TOFMS; 1472.1 (1472.5 calc for M+H).

43. DM-γ-PyImPy-γ-ImPyPy-β-PyPyPy-β-Dp:

The polyamide was prepared by machine assisted solid phase methods as awhite powder (13 mg, 52% recovery). UV (H2O) λ_(max) 242 nm, 310 nm(ε=75,000, calculated based on ε=8333/ring(30)); ¹H NMR (DMSO-d₆): δ10.28 (s, 1 H); 10.26 (s, 1 H), 9.99 (s, 1 H), 9.96 (s, 1 H), 9.94 (s, 1H), 9.90 (m, 3 H), 9.88 (s, 1 H), 9.3 (br s, 1 H), 9.2 (br s, 1 H), 8.05(m, 4 H), 7.52 (s, 1 H); 7.43 (s, 1 H); 7.27 (d, 1 H, J=1.6 Hz), 7.24(d, 1 H, J=1.7 Hz), 7.20 (d, 1 H, J=1.6 Hz), 7.17 (m, 4 H), 7.14 (d, 1H, J=1.6 Hz), 7.12 (d, 1 H, J=1.5 Hz), 7.03 (d, 1 H, J=1.6 Hz), 6.96 (d,1 H, J=1.6 Hz), 6.90 (d, 1 H, J=1.5 Hz), 6.86 (m, 2 H), 3.94 (s, 3 H),3.92 (s, 3 H), 3.81 (m, 12 H), 3.78 (m, 9 H), 3.56 (m, 4 H), 3.39 (m, 6H), 2.95 (m, 4 H), 2.76 (d, 6 H, J=4.6 Hz), 2.71 (d, 6 H, J=4.6 Hz),2.30 (m, 4 H), 1.88 (m, 2 H), 1.73 (m, 4 H). MALDI-TOF MS; 1543.3(1543.6 calc for M+H).

44. DM-γ-ImPyPy-γ-ImPyPy-β-PyPyPy-β-PyPyPy-β-Dp:

The polyamide was prepared by machine assisted solid phase methods as awhite powder (3 mg, 10% recovery). UV (H2O) λ_(max) 239 nm, 308 nm(ε=100,000, calculated based on ε=8333/ring); MALDI-TOF MS; 1981.3(1981.1 calc for M+H).

45. ImPyPyPy-γ-ImPyPyPy-β-Dp:

Polyamide ImPyPyPy-γ-ImPyPyPy-β-Dp was prepared by machine-assistedsolid phase methods as a white powder (17 mg, 56% recovery). HPLC, r.t.:26.1 min; UV, λ_(max) (ε): 234 nm (39,300), 312 nm (53,200); ¹H NMR(DMSO-d₆): δ10.53 (s, 1H), 10.27 (s, 1H), 10.04 (s, 1H), 9.96 (s, 1H),9.94 (s, 1H), 9.2 (br s, 1H), 8.08 (m, 3H), 7.49 (s, 2H), 7.44 (s, 1H),7.31 (d, 1H, J=1.0 Hz), 7.23 (d, 1 H, J=1.1 Hz), 7.19 (m, 3H), 7.10 (s,1H), 6.92 (d, 1 H, J=1.1 Hz), 6.90 (d, 1 H, J=1.1 Hz. 4.01 (s, 3H), 3.97 (s, 3H), 3.86 (m, 6H), 3.82 (m, 6H), 3.41 (q, 2 H, J=6.0 Hz), 3.22(q, 2 H, J=5.9 Hz), 3 .13 (q, 2 H, J=5.9 Hz), 3.0 (q, 2 H, J=5.6 Hz),2.76 (d, 6 H, J=4.8 Hz), 2.37 (m, 4H), 1.78 (m, 4H); MALDI-TOF MS:1223.4 (1223.3 calc. for M+H).

46. ImPyPyPy-γ-PyPyPyPy-β-Dp:

The polyamide ImPyPyPy-γ-PyPyPyPy-β-Dp was prepared by machine-assistedsolid phase methods as a white powder (12 mg, 19% recovery). HPLC, r.t.:29.5 min; UV, λ_(max) (ε): 238 nm (53,900), 312 nm (71,100); ¹H NMR(DMSO-d₆): δ10.46 (s, 1H), 10.24 (s, 1H), 9.96 (s, 1H), 9.90 (m, 5H),9.2 (br s, 1H), 8.25 (m, 1H), 8.00 (m, 3H), 7.44 (s, 1H), 7.39 (s, 1H),7.26 (d, 1 H, J=1.3 Hz), 7.24 (d, 1 H, J=1.5 Hz), 7.20 (m, 2H), 7 .16(m, 2H), 7.13 (m, 2 H), 7.11 (d, 1 H, J=1.4 Hz), 7.05 (d, 1 H, J=1.4Hz), 7.03 (d, 1 H, J=1.5 HZ), 6.93 (d, 1 H, J=1.3 Hz), 6.87 (m, 2H),6.84 (d, 1 H, J=1.5 Hz), 3.97 (s, 3H), 3.92 (s, 3H), 3.82 (m, 9H), 3.79(m, 6H), 3.76 (m, 6H), 3.73 (m, 2 H), 3.44 (q, 2 H, J=5.0 Hz), 3.17 (m,4H), 3.03 (m, 2H), 2.74 (d, 6 H, J=4.8 Hz), 2.50 (m, 2H) 2.33 (t, 2 H,J=6.7 Hz), 1.77 (m, 4H); MALDI-TOF MS: 1222.3 (1222.3 calc for M+H).

47. ImImImPy-γ-PyPyPyPy-β-Dp:

The product was synthesized by manual solid phase protocols andrecovered as a white powder (7.6 mg, 11% recovery). UV λ_(mix , 248)(42,000), 312 (48,500); ¹H NMR (DMSO-d₆) d 10.32 (s, 1H), 10.13 (s, 1H)9.93 (s, 1H), 9.90 (s, 1H), 9.89 (s, 1H), 9.84 (s, 1H), 9.59 (s, 1H),9.23 (br s, 1H), 8.09 (t, 1 H, J=5.3 Hz), 8.04 (m, 2H), 7.65 (s, 1H),7.57 (s, 1H), 7.46 (d, 1 H, J=0.6 Hz) 7.22 (m, 3H), 7.16 (s, 2H), 7.09(d, 1 H, J=0.8 Hz), 7.06 (d, 2 H, J=1.1 Hz), 7.00 (d, 1 H, J=1.7), 6.88(d, 1 H, J=1.8), 6.87 (d, 1 H, J=1.8 Hz), 4.02 (s, 3H), 4.00 (s, 3H),3.99 (s, 3H), 3.84 (s, 3H), 3.83 (s, 3H), 3.83 (s, 3H), 3.80 (s, 3H),3.79 (s, 3H), 3.37 (q, 2 H, J=6.2 Hz), 3.21 (q, 2 H, J=6 .4 Hz), 3.10(q, 2 H, J=6.2 Hz), 3.00 (m, 2H), 2.73 (d, 6 H, J=4.9 Hz), 2.34 (t, 2 H,J=7.2 Hz), 2.28 (t, 2 H, J=7.0 Hz), 1.76 (m, 4H); MALDI-TOF-MS, 1225.9(1224.3 calc. for M+H).

48. ImImImPy-γ-PyPyPyPy-β-Dp-NH₂:

A sample of polyimide machine-synthesized on resin (350 mg, 0.16mmol/gram) was placed in a 20 mL glass scintillation vial, and treatedwith 2 mL 3,3′-diamino-N-methyldipropylamine at 55° C. for 18 hours. Theresin was removed by filtration through a disposable propylene filter,and the resulting solution dissolved with water to a total volume of 8mL, and purified directly by preparatory reversed phase HPLC to provideImImImPy-γ-PyPyPyPy-β-Dp-NH₂ (31 mg, 40% recovery) as a white powder. ¹HNMR (DMSO-d₆) δ10.37 (s, 1H), 10.16 (s, 1H), 9.95 (s, 1H), 9.93 (s, 1H),9.91 (s, 1H), 9.86 (s, 1H), 9.49 (br s, 1H), 9.47 (s, 1H), 8.12 (m, 3H),8.0 (br s , 3H), 7.65 (s, 1H), 7.57 (s, 1H), 7.46 (s, 1H), 7.20 (m, 3H),7 .16 (m, 2H), 7.09 (d, 1 H, J=1.5 Hz), 7.05 (m, 2H), 7.00 (d, 1 H,J=1.6 Hz), 6.88 (m, 2H), 4.01 (s, 3H), 3.99 (s, 3H), 3.98 (s, 3H), 3.83(s, 3H), 3.82 (s, 3H), 3.81 (s, 3H), 3.79 (s, 3H), 3.78 (s, 3H), 3.36(q, 2 H, J=5.3 Hz), 3.21-3.05 (m, 8H), 2.85 (q, 2 H, J=4.9 Hz), 2.71 (d,3 H, J=4.4 Hz), 2.34 (t, 2 H, J=5.9 Hz), 2.26 (t, 2 H, J=5.9 Hz), 1.85(quintet, J=5.7 Hz), 1.72 (m, 4H). MALDI-TOF-MS, 1267.1 (1267.4 calc.For M+H).

49. ImImImPy-γ-PyPyPyPy-β-DP-EDTA:

Compound was prepared as described for ImImIm-γ-PyPyPy-(β-Dp-EDTA. (3.8mg, 40%). ¹H NMR (DMSO-d₆) δ10.34 (s, 1H), 10.11 (s, 1H), 9.92 (s, 1H),9.90 (s, 1H), 9.89 (s, 1H), 9.84 (s, 1H), 9.57 (s, 1H), 8.42 (m, 1H),8.03 (m, 3H), 7.64 (s, 1H), 7.56 (s, 1H), 7.44 (s, 1H), 7.20 (m, 3H),7.15 (m, 2H), 7.07 (d, 1 H, J=1,6 Hz), 7.05 (m, 2H), 6.99 (d, 1 H, J=1.6Hz), 6.87 (m, 2H), 4.00 (s, 3H), 3.98 (s, 3H), 3.97 (s, 3H), 3.83 (m,4H), 3.82 (s, 6 H), 3.79 (s, 3H), 3.78 (s, 6H), 3.67 (m, 4H), 3.4-3.0(m, 16H), 2.71 (d, 3 H, J=4.2 Hz), 2.34 (t, 2 H, J=5.4 Hz), 2.25 (t, 2H, J=5.9 Hz), 1.72 (m, 6H). MALDI-TOF-MS, 1542.2 (1542.6 calc. for M+H).The polyimide was loaded with Fe (II) by standard methods.

50. ImImPyPy-γ-ImImPyPy-β-Dp:

The polyimide ImImPyPy-γ-ImImPyPy-β-PAM-Resin was assembled on 0.2mmol/gram Box-β-PAM-resin by machine assisted synthesis. The γ-Im stepwas assembled using Boc-γ-Im, acid (HBTU, DIEA), all other residues wasadded as appropriate activated Boc protected monomer units. A sample ofresin (250 mg, 0.16 mmol/gram²¹) was placed in a 20 mL glassscintillation vial, 2 mL dimethylaminopropylamine added and the mixtureallowed to stand at 55° C. for 18 hours. Resin was removed by filtrationthrough a disposable propylene filter, and the resulting solutiondiluted with water to a total volume of e mL, and purified directly bypreparatory reversed phase HPLC to provide ImImPyPy-γImImPyPy-β-Dp (26mg, 45% recovery) as a white powder. UV λ_(max)(H₂O) 248, 312 (66,000);¹H NMR (DMSO-d₆) d 10.34 (m, 2 H); 10:32 (m, 2H); 9.73 (m, 2H); 9.5 (brs, 1H), 9.32 (s, 1H); 8.10 (m, 3H); 7.55 (m, 2H); 7.52 (s, 1H); 7.44 (s,1H); 7.23 (m, 2H), 7.14 (m, 4H); 7.06 (d, 1 H, J=1.4 Hz); 6.86 (m, 2H);3.98 (m, 9H); 3.95 (s, 3H); 3.81 (m, 6H); 3.77 (m, 6H); 3.31 (m, 2H);3.17 (t, 2 H, J=5.5 Hz) 3.06 (m, 2 H, J=5.7 Hz); 2.93 (m, 2 H, J=4.7Hz); 2.74 (d, 6 H, J=4.4 Hz); 2.30 (m, 4H); 1.74 (m, 4H); MALDI-TOF-MS,1224.9 (1225.3 calc. for M+H).

51. ImPyImPy-γ-ImPyImPy-β-Dp:

The polyamide ImPyImPy-γ-ImPyImPy-β-PAM-Resin was assembled on 0.2mmol/gram Boc-β-PAM-resin by manual polyamide synthesis. The Py-Im andγ-Im steps were addedusing Boc-λ-Im acid and Boc-Py-Im acid (HBTU,DIEA), all other residues were added as appropriate activated Bocprotected monomer units. A sample of resin (250 mg, 0.16 mmol/gram²¹)was placed in a 20 mL glass scintillation vial, 2 mLdimethylaminopropylamine added and the mixture allowed to stand at 55°C. for 18 hours. Resin was removed by filtration through a disposablepropylene filter, and the resulting solution diluted with water to atotal volume of 8 mL, and purified directly by preparatory reversedphase HPLC to provide ImPyImPy-γ-ImPyImPy-β-Dp (19 mg, 32% recovery) asa white powder. UV λ_(max)(H₂O) 246, 312 (66,000); ¹H NMR (DMSO-d₆) d10.33 (m, 2H); 10.25 (m, 2H); 10.04 (m, 2H) ; 9.95 (s, 1H); 9.5 (br s,1H), 8.10 (m, 3H); 7.57 (m, 2H); 7.48 (s, 1H); 7.42 (s, 1H); 7,40 (s,1H); 7.23 (m, 2H), 7.17 (d, 1 H; J=1.5 Hz); 7.03 (d, 1 H, J=1.5 Hz);6.98 (m, 3H); 4.02 (s, 3H); 3.99 (m, 6H); 3.81 (m, 6H); 3.97 (s, 3H);3.88 (m, 6H); 3.83 (m, 6H); 3.42 (m, 2H); 3.18 (t, 2 H, J=5.2 Hz) 3.06(m, 2 H, J=5.5 Hz); 2.80 (m, 2 H, J=4.7 Hz); 2.76 (d, 6 H, J=4.4 Hz),2.38 (m, 4H); 1.93 (m, 4H); MALDI-TOF-MS, 1225.2. (1225.3 calc. forM+H).

52. ImImImIm-γ-PyPyPyPy-β-Dp:

The polyamide ImImImIm-γ-PyPyPyPy-β-PAM-Resin was assembled on 0.2mmol/gram Boc-b-PAM-resin by manual polyamide synthesis. The γ-Im stepwas added using Boc-γ-Im acid (HBTU, DIEA), all other residues wereadded as appropriate activated Boc protected monomer units. A sample ofresin (150 mg, 0.16 mmol/gram²¹) was placed in a 20 mL glassscintillation vial, 2 mL dimethylaminopropylamine added and the mixtureallowed to stand at 55° C. for 18 hours. Resin was removed by filtrationthrough a disposable propylene filter, and the resulting solutiondiluted with water to a total volume of e mL, and purified directly bypreparatory reversed phase HPLC to provide ImImImIm-γ-PyPyPyPy-β-Dp (12mg, 21% recovery) as a white powder. UV λ_(max)(H₂O) 246, 314 (66,000);¹H NMR (DMSO-d₆) d 9.91 (m, 2 H); 9.89 (m, 4H); 9.83 (s, 1H); 9.60 (s,1H); 9.5 (br s, 1H); 8.34 (m, 1H); 8.10 (m, 2H); 7.63 (m, 2H); 7.50 (s,1H); 7.42 (s, 1H); 7.19 (m, 2H); 7.13 (m, 2H); 7.04 (m, 2H); 6.86 (m,2H); 1.98 (m, 6H); 3.96 (s, 3H); 3.93 (s, 3H); 3.81 (m, 6H); 3.77 (s,3H); 3.73 (s, 3H); 3.30 (m, 2H); 3.10 (t, 2 H, J=5.7 Hz), 3.09 (m, 2 H,J=5.5 Hz), 2.91 (m, 2 H, J=4.6 Hz), 2.71 (d, 6 H, J=4.2 Hz), 2.32 (m,4H); 1.70 (m, 4H); MALDI-TOF-MS, 1225.6 (1225.3 calc. for M+H).

53. ImImImPy-β-PyPyPyPy-β-DP:

A sample of ImImImPy-β-PyPyPyPy-β-resin prepared by machine-assistedsolid phase synthesis 1240 mg, 0.16 mmol/gram) was placed in a 20 mLglass scintillation vial, and treated with dimethylaminopropylamine (2mL) at 55° C. for 18 hours. Resin was removed by filtration, and thefiltrate diluted to a total volume of 8 mL with 0.1% (wt/v) aqueous TFA.The resulting crude polyamide/amine solution was purified directly byreversed phase HPLC to provide the trifluoroacetate salt ofImImImPy-β-PyPyPyPy-β-Dp (31 mg, 40% recovery) as a white powder. ¹H NMR(300 MHz, [D₆] DMSO, 20° C.): d=10.37 (s, 1H; NH), 10.12 (s, 1H; NH),9.95 (s, 1H; NH), 9.94 (s, 1H; NH), 9.93 (s, ¹H; NH), 9.92 (s, 1H; NH),9.59 (s, 1H; NH), 9.4 (br s, 1H; CF₃COOH), 8.09 (m 3H; NH), 7.65 (s, 1H;CH), 7,56 (s, 1H; CH), 7,45 (s, 1H; CH), 7.27 (d, ²J(H,H)=1.3 Hz, 1H;CH), 7.22 (m, 2H; CH), 7.18 (d, ²J(H,H)=1.3 Hz, 1H; CH), 7.16 (d,²J(H,H)=1.0 Hz, 1H; CH), 7.07 (m, 2H; CH;, 6.95 (d, ²J(H,H)=1.1 Hz, 1H;CH), 6.88 (d, ²J(H, H)=1.4 Hz, 1H; CH), 6.86 (d, ²J(H, H)=1.3 Hz, 1H;CH), 4.01 (s, 3H; NCH₃), 3.98 (m, 2H; NCH₃), 3.83 (s, 3H; NCH₃), 3.82(m, 6H; NCH ₃), 3.80 (s, 3H; NCH₃), 3.78 (s, 3H; NCH₃), 3.4 (m, 6H;CH₂), 3.11 (q, ⁴J(H,H)=5.2 Hz, 2H; CH₂), 2.94 (q, ⁴J (H, H). 5.3 Hz, 2H;CH₂), 2.69 (d, ²J(H,H)=4.4 Hz, 6H; N(CH₃)₂), 2.33 (t, ³J (H,H)=5.4 Hz,2H; CH₂), 1.75 (q, ⁵J(H,H)=7.1 Hz, 2H; CH₂); UV/VIS (H₂O) λ_(max)(q)=304 (66,600, calculated from ε=8,333/ring^((14c))), 241 nm;MALDI-TOF-MS [M⁺−H] 1210.4: calc. 1210.3.

54. ImImPyPy-β-PyPyPypy-β-Dp:

A sample of ImImPyPy-β-PyPyPyPy-β-resin prepared by machine-assistedsolid phase synthesis (240 mg, 0.16 mmol/gram¹⁵) was placed in a 20 mLglass scintillation vial, and treated with dimethylaminopropylamine (2mL) at 55° C. for 18 hours, Resin was removed by filtration, and thefiltrate diluted to a total volume of 8 mL with 0.1% (wt/v) aqueous TFA.The resulting crude polyamide/amine solution was purified directly byreversed phase HPLC to provide the trifluoroacetate salt ofImImPyPy-β-PyPyPyPy-β-Dp (31 mg, 40% recovery) as a white powder. ¹H NMR(300 MHz, [D_(6] DMSO,) 20° C. δ=10.38 (s, 1H; NH), 9.95 (s, 1H; NH),9.93 (s, 1H; NH), 9.91 (s, 1H; NH), 9.90 (m, 2H; NH), 9.76 (s, 1H; NH),9.4 (br s, 1H; CF₃COOH), 8.09 (m, )H; NH), 7.56 (s, 1H; CH), 7,46 (s,1H; CH), 7.27 (d, ²J(H,H)=1.8 Hz, 1H; CH), 7,21 (d, ²J(H,H)=1.7 Hz, 1H;CH), 7.20 (d, ²J(H,H)=1.9 Hz, 1H; CH), 7.19 (d, ²J(H,H)=1.9 Hz, 1H; CH),7.16 (d, ²J(H,H)=1.9 Hz, 1H; CH), 7.15 (d, ²J(H,H)=1.6 Hz, 1H; CH), 7,14(d, ²J(H,H)=1.9 Hz, 1H; CH), 7.12 (d, ²J(H,H)=1.6 Hz, 1H; CH), 7.07 (s,1H; CH), 7.05 (d, ²J(H,H)=1.5 Hz, 1H; CH), 6.87 (d, ²J(H,H)=1.9 Hz, 1H;CH), 6.86 (d, ³J(H,H)=1.6 Hz, 1H: CH), 6.84 (d, ²J(H,H)=1.6 Hz, 1H; CH),3.99 (m, 6H; NCH₃), 3.82 (m, 12H; NCH₃), 3.80 (s, 3H; NCH₃), 3.78 (s,3H; NCH₃), 3.4 (m, 6H; CH₂), 3.09 (q, ⁴J(H,H)=5.6 Hz, 2H; CH₂), 2.97 (q,⁴J(H,H)=5.2 Hz, 2H; CH₂), 2.71 (d, ²J(H,H)=4.2 Hz, 6H; N(CH₃)₂), 2:32(t, ³J(H,H)=5.1 Hz, 2H; CH₂), 1.71 (q, ⁵J(H,H)=7.4 Hz, 2H; CH₂); UV/VIS(H₂O) λ_(max) (ε)=306 (66,600, calculated from e=8,333/ring), 243 nm;MALDI-TOF-MS (M⁺−H) 1209.1; calc. 1209.3.

55. ImPyPyPy-βPyPyPyPy-βDp:

A sample of ImPyPyPy-β-PyPyPyPy-β-resin prepared by machine-assistedsolid phase synthesis 1240 mg, 0.16 mmol/gram) was placed in a 20 mLglass scintillation vial, and treated with dimethylaminopropylamine (2mL) ac 55° C. for 18 hours. Resin was removed by filtration, and thefiltrate diluted to a total volume of 8 mL with ).1% (wt/v) aqueous TFA,The resulting crude polyamide/amine solution was purified directly byreversed phase HPLC to provide the trifluoroacetate salt ofImPyPyPy-β-PyPyPyPy-β-Dp (31 mg, 40% recovery) as a white powder. ¹H NMR(300 MHz, [D₆] DMSO, 20° C. δ=10.49 (s, 1H; NH), 9.97 (s, 1H; NH), 9.95(s, 1H; NH), 9.94 (s, 1H; NH), 9.93 (m, 2H; NH), 9.91 (s, 1H; NH), 9.4(br s, 1H; CF₃COOH), 8.10 (m, 3H; NH), 7.38 (s, 1H; CH), 7.28 (d, ²J (H,H)=1.6 Hz, 1H; CH), 7.22 (m, 3H; CH), 7.19 (m, 2H; CH), 7.16 (m, 2H;CH), 7.09 (m, 2H; CH), 7.04 (m, 2H; CH), 6.87 (d, ²J(H,H)=1.6 Hz, 1H;CH), 6.86 (d, ²J(H,H)=1.6 Hz, 1H; CH), 6.84 (d, ²J(H,H)=1.5 Hz, 1H; CH),3.97 (s, 3H; NCH₃), 3.82 (m, 15H; NCH₃), 3.80 (s, 3H; NCH₃), 3.78 (s,3H; NCH₃), 3.4 (m, 6H; CH₂), 3.10 (q, ⁴J(H;H)=5.4 Hz, 2H; CH₂), 2.98 (q,⁴J(H,H)=5.3 Hz, 2H; CH₂), 2.72 (d, ²J(H,H)=4.7 Hz, 6H; N(CH₃)₂), 2.33(t, ³J(H,H)=7.0 Hz, 2H; CH₂), 1.71 (q, ⁵J(H,H)=6.4 Hz, 2H; CH₂), UV/VIS(H₂O) λ_(max) (υ)=312 (66,600, calculated from ε=8,333/ring), 244 nm;MALDI-TOF-MS [M⁺−H] 1208.2: calc. 1208.3.

56. ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp:

A sample of ImPyPyPyPy-γ-ImPyPyPyPy-β-resin prepared by machine-assistedsolid phase synthesis (240 mg, 0.16 mmol/gram) was placed in a 20 mLglass scintillation vial, and treated with dimethylaminopropylamine (2mL) at 55° C. for 18 hours. Resin was removed by filtration, and thefiltrate diluted to a total volume of e mL with 0.1% (wt/v) aqueous TFA.The resulting crude polyamide/amine solution was purified directly byreversed phase HPLC to provide the trifluoroacetate salt ofImPyPyPyPy-γ-ImPyPyPyPy-β-Dp (13 mg, 18% recovery) as a white powder. UV(H₂O) λ_(max) 241, 316 (ε) 83300 (calculated based on ε=8,333/ring); ¹HNMR (DMSO-d₆) δ 10.52 (s, 1H), 10.29 (s, 1 H), 10.04 (s, 1H), 10.00 (s,1H), 9.97 (m, 3H), 9.92 (m, 3H), 9.22 (br s, 1H), 8.06 (m, 3H), 8.03 (m,2H), 7.46 (s, 1H), 7.41 (s, 1H), 7.29 (d, 1 H, J=1.0 Hz), 7.23 (m, 1H),7.17 (m, 1H), 7.07 (m, 1H), 6.90 (d, 1 H, J=6.9 Hz), 3.99 (s, 3H), 3.94(s, 3H), 3.85 (m, 6H), 3.79 (s, 3H), 3.38 (q, 2 H, J=3.2 Hz), 3.20 (q, 2H, J=2,7 Hz), 3.11 (q, 2 H, J=1.8 Hz), 3.00 (q, 2 H, J=2 .1 Hz), 2.72(d, 6 H, J+4.8 Hz), 2.35 (m, 4H), 1.75 (m, 4H); MALDI-TOF-MS, 1466.1(1467.6 calc. for M+H).

57. ImImPyPyPy-γ-ImPyPyPyPy-β-Dp:

The polyamide was prepared as described for ImPyPyPyPy-γ-ImPyPyPyPy-β-Dpas a white powder (28 mg, 34% recovery). UV λ_(max) 310 (83,300); ¹H;NMR (DMSO-d₆) d 10.39 (s, 1H), 10.28 (s, 1H), 10.02 (s, 1H), 9.99 (s,1H), 9.96 (m, 2H), 9.91 (s, 2H), 9,76 (s, 1H), 9.18 (br s, 1H), 8.05 (m,3H), 7.57 (s, 1H), 7.46 (s, 2H), 7.25 (dd, 2 H, J=5.6), 7.23 (m, 4H),7.16 (m, 4H), 7.07 (m, 4H), 6.88 (d, 1 H, J=5.1), 4.00 (s, 3H), 3.94 (s,3H), 3.85 (m, 6H), 3.79 (s, 3H), 2.99 (q, 2 H, J=5.1), 2.73 (d, 6 H,J=4.8 Hz), 2.34 (m, 4H), 1.75 (m, 4H); MALDI-TOF-MS, 1468.2 (1468.6calc. fox M+H).

58. ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp-NH₂:

A sample of ImPyPyPyPy-γ-ImPyPyPyPy-β-resin prepared by machine-assistedsolid phase synthesis (240 mg, 0.16 mmol/gram) was placed in a 20 mLglass scintillation vial, and treated with3,3-diamino-N-methyldipropylamine (2 mL) at 55° C. for 18 hours. Resinwas removed by filtration, and the filtrate diluted to a total volume of8 mL with 0.1% (wt/v) aqueous TFA. The resulting crude polyamide/aminesolution was purified directly by reversed phase HPLC to provide thetrifluoroacetate salt of ImPyPyPyPy-γ-ImPyPyPyPy-β-NH₂ (31 mg, 40%recovery) as a white powder. UV λ_(max) 241, 316 (ε) 83300 (calculatedbased on ε=8,333/ring⁵); ¹H NMR (DMSO-d₆) δ10.53 (a, 1H), 10.26 (s, 1H), 10.03 (s, 1H), 10.00 (s, 1H), 9.96 (m, 2H), 9.92 (m, 2H), 9.6 (br s,1H), 8.07 (m, 4H), 7.89 (s, 3H), 7 .45 (s, 1H), 7.41 (s, 1H), 7.27 (d, 2H, J=8.5 Hz), 7.23 (m, 4H), 7.16 (m, 4H), 7.06 (m, 4H), 6.87 (m, 2H),3.98, (s, 3H); 3.94 (s, 3H), 3.84, (m, 6H), 3.79 (s, 3H), 3.35 (q, 2 H,J=5.7 Hz), 3.16 (m, 8H), 2.85 (q, 2 H, J=5.6 Hz), 2.72 (d, 2 H, J=4.2Hz), 2.34 (m, 2H), 1.91 (m, 4H), 1.78 (m, 4H). MALDI-TOF MS, 1510.4(1510.7 calc. for M+H).

59. ImImPyPyPy-γ-ImPyPyPyPy-β-Dp-NH₂:

The polymide was prepared as a white powder as described forImPyPyPyPy-γ-ImPyPyPyPy-β-NH₂. ¹H NMR (DMSO-d₆) δ10.39 (s, 1H), 10.28(s, 1H), 10.03 (s, 1H), 10.03 (s, 1H), 9.92 (m, 2H), 9.82 (s, 1H), 9.66(br s, 1H), 8.11 (m, 4H), 7.89 (s, 3H), 7.57 (s, 1H), 7.46 (d, 2 H,J=2.4 Hz), 7.27 (dd, 2 H, J=1.0 Hz), 7.23 (m, 4H), 7.16 (m, 4H), 7.08(m, 4H), 6.88 (m, 1H), 4,00 (s, 3H), 3.94 (s, 3H), 3.78 (s, 3H), 3.19(q, 2 H, J=5.1 Hz), 3.05 (m, 8H), 2.86 (q, 2 H, J=4.8 Hz), 2.72 (d, 2 H,J=4.4 Hz), 2.34 (m, 4H), 1.90 (m, 4H), 1.78 (m, 4H), MALDI-TOF-MS,1510.4 (1511.7 calc. for M+H).

60. ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp-EDTA:

EDTA-dianhydride (50 mg) was dissolved by heating at 55° C. for 5 min.in a solution of DMSO/NMP (1 ml) and DIEA (1 mL). The dianhydridesolution was added to ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp-NH₂ (8.1 mg)dissolved in DMSO (750 μL) The mixture was heated at 55° C. for 25minutes, and treated with 0.1M NaOH (3 mL), and heated at 55° C. for 10minutes. Aqueous 0.1% (wt/v) TFA was added to adjust the total volume to8 mL and the solution purified directly by preparatory HPLCchromatography to provide ImPypypypy-γ-ImPyPyPyPy-β-Dp-EDTA as a whitepowder. (2.4 mg, 22% recovery) MALDI-TOF-MS, 1766.4 (1766.7 calc. forM+H).

E. Plasmids, Footprinting, Affinity Cleavage

1. Construction of plasmid DNA

The experimental target plasmid pSES9hp was constructed by hybridizationof the inserts:5′-GATCCTATGTCAGTCATGCGGATGACTGTCAGTCATGGCCATGACTGTCAGTCATGCGCATGACTGTCAGTCTTAAGC-3′and5-GATACAGTCAGTACCCCTACTGACAGTCAGTACCGGTACTGACAGTCAGTACGCGTACTGACAGTCAGAATTCGTCGA-3′.

The hybridized insert was ligated into linearized pUC19 BamHI/HindIIIplasmid using T4 DNA ligase. The resultant constructs were used totransform Top10F′ OneShot competent cells from Invitrogen.Ampicillin-resistant white colonies were selected from 25 mLLuria-Bertani medium agar plates containing 50 μg/mL ampicillin andtreated with XGAL and IPTG solutions. Large-scale plasmid purificationwas performed with Qiagen Maxi purification kits. Dideoxy sequencing wasused to verify the presence of the desired insert. Concentration of theprepared plasmid was determined at 260 nm using the relationship of 1 ODunit=50 μg/mL duplex DNA.

2. Preparation of 3′-and 5′-End-Labeled Restriction Fragments

The plasmid pSES9hp was linearized with EcoRI and PvuII and then treatedwith Klenow fragment, deoxyadenosine 5′[α-³²P] triphosphate andthymidine 5′-[α-³²P] triphosphate for 3′ labeling. Alternatively,pSES9hp was linearized with EcoRI, treated with calf alkalinephosphatase, and then 5′ labeled with T4 polynucleotide kinase anddeoxyadenosine 5′-[γ-³²P]triphosphate. The 5′labeled fragment was thendigested with PvuII. The labeled fragment (3′ or 5′) was loaded onto a5% non-denaturing polyacrylamide gel, and the desired 282 base pair bandwas visualized by autoradiography and isolated. Chemical sequencingreactions were performed according to published methods. (Maxam, A. M. &Gilbert, W. S. (1980). Sequencing End-Labeled DNA with Base-SpecificChemical Cleavages. Methods Enzymol. 65, 499-560; Iverson, H. L. &Dervan, P. B. (1987). Adenine-specific DNA chemical sequencing reaction.Methods Enzymol. 15, 7823-7830.)

3. MPE•Fe(II) Footprinting

All reactions were carried out in a volume of 40 μL. A polyamide stocksolution or water for reference lanes) was added to an assay bufferwhere the final concentrations were: 25 mM Tris-acetate buffer (pH 7.0),10 mM NaCl, 100 μM/base pair calf thymus DNA, and 30 kcpm 3′- or5′-radiolabeled DNA. The solutions were allowed to equilibrate for 4hours. A fresh 50 μm MPE•Fe(II) solution was made from 100 μL of a 100μm MPE solution and 100 μL of a 100 μM ferrous ammonium sulfate (Fe(NH₄)₂(S0 ₄)₂•6H₂O) solution. MPE•Fe(II) solution (5 μM) was added tothe equilibrated DNA, and the reactions were allowed to equilibrate for5 minutes. Cleavage was initiated by the addition of dithiothreitol (5mM) and allowed to proceed for 14 min. Reactions were stopped by ethanolprecipitation, resuspended in 100 mM tris-Borate-EDTA/80% formamideloading buffer, denatured at 85° C. for 5 min, placed on ice, and halfof each tube (−15 kcpm) was immediately loaded onto an 8% denaturingpolyacrylamide gel (5% crosslink, 7 M urea) at 2000 V.

4. Affinity Cleaving.

All reactions were carried out in a volume of 40 μL. A polyamide stocksolution or water (for reference lanes) was added to an assay bufferwhere the final concentrations were: 25 mM Tris-acetate buffer (pH 7.0),10 mM NaCl, 100 μM/base pair calf thymus DNA, and 20 kcpm 3′- or5′-radiolabeled DNA. The solutions were allowed to equilibrate for 4hours. A fresh solution of ferrous ammonium sulfate(Fe(NH₄)₂(SO₄)₂.6H₂O) (10 μM) was added to the equilibrated DNA, and thereactions were allowed to equilibrate for 15 minutes. Cleavage wasinitiated by the addition of dithiothreitol (10 mM) and allowed toproceed for 30 min. Reactions were stopped by ethanol precipitation,resuspended in 100 mM tris-borate-EDTA/80% formamide loading buffer,denatured at 85° C. for 5 min, placed on ice, and the entire sample wasimmediately loaded onto an 8% denaturing polyacrylamide gel (5%crosslink, 7 M urea) at 2000 V.

5. Identification of Binding Orientation by Affinity Cleaving.

Affinity cleavage assays (25 mM Tris-acetate, 10 mM NaCl, 100 μM/basepair calf thymus DNA, pH 7.0 and 22° C.) were performed in order toidentify the binding orientations of the EDTA analogues of the threehairpin polyamides: ImImPyPy-γ-ImImPyPy-β-Dp-EDTA,ImPyImPy-γ-ImPyImPy-β-Dp-EDTA, and ImImImIm-γ-PyPyPyPy-β-Dp-EDTA Thepolyamides ImImPyPy-γ-ImImPyPy-β-Dp-EDTA, ImPyImPy-γ-ImPyImPy-β-Dp-EDTArecognize their respective palindromic match sequences, 5′-TGGCCA-3′ and5′-TGCGCA-3′, in two equivalent orientations, consistent with hairpinformation. In contrast, the polyamide ImImImIm-γ-PyPyPyPy-β-Dp-EDTArecognizes a non-palindromic sequence, 5′-TGGGGA-3′, in a singleorientation with cleavage visible only on the 5′-side of the site, aspredicted by the hairpin model.

Depicted in FIG. 17 is a representative affinity cleaving experiment ona 3′-³²P-labeled 282 bp EcoRI/PvuII restriction fragment from plasmidpSES9hp. The 5′-TGGCCA-3′, 5′-TGCGCA-3 and 5′-TGGGGA-3′ sites are shownon the right side of the autoradiogram. Lane 1, A reaction; lane 2, Creaction; lanes 3-5, 1 μM, 2 μM and 5 μM ImImPyPy-γ-ImImPyPy-β-Dp-EDTA(1-E); lanes 6-8, 1 μM, 2 μM and 5 μM ImPyImPy-γ-ImPyImPy-β-Dp-EDTA(2-E); lanes 9-11, 1 μM, 2 μM and 5 μM ImImImIm-γ-PyPyPyPy-β-Dp-EDTA(3-E); lane 12, intact DNA. All lanes contain 15 kcpm 3′-radiolabeledDNA, 25 mM Tris-acetate buffer (pH 7.0), 10 mM NaCl, and 100 μM/basepair calf thymus DNA. (Right) Affinity cleavage patterns forImImPyPy-γ-ImImPyPy-β-Dp-EDTA and ImPyImPy-γ-ImPyImPy-β-Dp-EDTA at 1 μMconcentration, and ImImImIm-γ-PyPyPyPy-β-Dp-EDTA at 5 μM concentration.Illustration of the 282 bp restriction fragment with the position of thesequence indicated. Bar heights are proportional to the relativeprotection from cleavage at each band. Boxes represent equilibriumbinding sites determined by the published model, and only sites thatwere quantitated by DNase I footprint titrations are boxed.

6. DNase I Footprinting

All reactions were carried out in a volume of 400 μL. We note explicitlythat no carrier DNA was used in these reactions. A polyamide stocksolution or water (for reference lanes) was added to an assay bufferwhere the final concentrations were: 10 mM CaCl₂, and 20 kcpm3′-radiolabeled. DNA. The solutions were allowed to equilibrate for aminimum of 12 hours at 22° C. Cleavage was initiated by the addition of10 μL of a DNase I stock solution (diluted with 1 mM DTT to give a stockconcentration of 0.28 u/mL) and was allowed to proceed for 5 min at 22°C. The reactions were stopped by adding 50 mL of a solution containing2.25 M NaCl, 150 mM EDTA, 0.6 mg/mL glycogen, and 30 mM base-pair calfthymus DNA, and then ethanol precipitated. The cleavage products wereresuspended in 100 mM tris-borate-EDTA/80% formamide loading buffer,denatured at 85° C. for 5 min, placed on ice, and immediately loadedonto an 8% denaturing polyacrylamide gel (5% crosslink, 7 M urea) at2000 V for 1 hour. The gels were dried under vacuum at 80° C., thenquantitated using storage phosphor technology.

The data were analyzed by performing volume integrations of the5′-TGGCCA-3′, 5′-TGCGCA-3′, and 5′-TGGGGA-3′ sites and a reference site.The apparent DNA target site saturation θ_(app), was calculated for eachconcentration of polyamide using the following equation: $\begin{matrix}{\theta_{app} = {1 - \frac{I_{tot}/I_{ref}}{I_{tot}{{^\circ}/I_{ref}}{^\circ}}}} & (1)\end{matrix}$

where I_(tot) and I_(ref) are the integrated volumes of the target andreference sites, respectively, and I_(tot) ^(o) and I_(ref) ^(o)correspond to those values for a DNase I control lane to which nopolyamide has been added. The ([L]_(tot), θ_(app)) data points were fitto a Langmuir binding isotherm (eq 2, n=lor n=2) by minimizing thedifference between θ_(app) and θ_(fit), using the modified Hillequation: $\begin{matrix}{\theta_{fit} = {\theta_{\min} + {\left( {\theta_{\max} - \theta_{\min}} \right)\frac{{K_{a}^{n}\lbrack L\rbrack}_{tot}^{n}}{1 + {K_{a}^{n}\lbrack L\rbrack}_{tot}^{n}}}}} & (2)\end{matrix}$

where [L]_(tot) corresponds to the total polyamide concentration, K_(a)corresponds to the equilibrium association constant, and θ_(min) andθ_(max) represent the experimentally determined site saturation valueswhen the site is unoccupied or saturated, respectively. Data were fitusing a nonlinear least-squares fitting procedure of KaleidaGraphsoftware (version 2.1, Abelbeck software) with K_(a), θ_(max), andθ_(min) as the adjustable parameters. All acceptable fits had acorrelation coefficient of R>0.97. At least three sets of acceptabledata were used in determining each association constant. All lanes fromeach gel were used unless visual inspection revealed a data point to beobviously flawed relative to neighboring points. The data werenormalized using the following equation: $\begin{matrix}{\theta_{norm} = \frac{\theta_{app} - \theta_{\min}}{\theta_{\max} - \theta_{\min}}} & (3)\end{matrix}$

7. Quantitation by Storage Phosphor Technology Autoradiography

Photostimulable storage phosphorimaging plates (Kodak Storage PhosphorScreen S0230 obtained from Molecular Dynamics) were pressed flat againstgel samples and exposed in the dark at 22° C. for 12-20 h. A MolecularDynamics 400S PhosphorImager was used to obtain all data from thestorage screens. The data were analyzed by performing volumeintegrations of all bands using the ImageQuant v. 3.2.

Example 2 Synthesis and Oxidative Cleavage of Double-Helical Dna ByPolyamides Modified With A Polyamide-Ni(Ii) Tripeptide Conjugate

Many anticancer drugs act through their ability to modify DNA. Novelpolyamide conjugates have been designed which modify double-helical DNAin a sequence specific manner. More specifically the metalopeptideNi(II)•Gly-Gly-His has been shown to promote the efficient oxidativecleavage of DNA in the presence of monoperoxyphthalic acid. (Mack andDervan, J. Am. Chem. Soc., 112, 4604 (1990); Mack and Dervan,Biochemistry, 31, 9399 (1992)).

The reaction is thought to proceed through a mechanism that involvesabstraction of hydrogen atom(s) from the deoxyribose backbone of DNA bya nondiffusable high valent nickel bound oxygen. Bifunctional conjugateswere designed in order to combine the ability of polyamides to recognizeany predetermined DNA sequence with the Ni(II)•Gly-Gly-His chemistry.The symmetric anhydride of the amino acid His and the activated ester ofGly were coupled to the extended hairpin polyamide directly on theβ-alanine-Pam resin employing solid phase chemistry protocols.Denaturing polyacrylamide gel electrophoresis of 32P end-labeled DNAtreated with the Ni(II)•Gly-Gly-His modified polyamide at pH 7.5demonstrated the ability of the conjugate to cleave the double helicalDNA is a sequence selective manner in 77% and 72% yields on the3′-end-labeled DNA (at 10 nM polyamide). The chemical structure of theNi(II)•Gly-Gly-His modified polyamide is shown in FIG. 18.

Example 3 Sequence Specific Alkylation of Dna By Pyrrole-ImidazolePolyamides Modified With Dna Reactive Agents

The design of sequence specific DNA binding-modifying molecules requiresthe integration of two separate entities: recognition and functionalreactivity. The present inventor has discovered ligands which combinepyrrole-imidazole polyamide DNA binding motifs with mechanism basedreactive functionalities capable of electrophilic modification of basesin the minor groove.

The design of sequence specific molecules for alkylation of doublehelical DNA requires both a specific DNA binding molecule and an atomspecific DNA cleaving moiety. Hairpin polyamides are sequence specificmolecules that can bind to any predetermined DNA sequence. Bromoacetyland the prodrug analogue of the cyclopropyl electrophile of CC-1065react in an atom specific manner with double helical DNA. By tethering abromoacetyl moiety or the prodrug analogue of the cyclopropylelectrophile of CC-1065 to a hairpin polyamide the present inventor hasdiscovered a sequence specific DNA alkylating agent which can betargeted to any predetermined DNA sequence at subnanomolarconcentration.

The two criteria for successful bifunctional molecule design aresequence specific reactions at designated single atoms within the boundcomplex, and cleavage yields that are quantitative under physiologicalconditions (i.e. neutral pH, 37° C., 100-200 nM KCl/NaCl). In order tomaximize stoichiometric reaction on the DNA, the ‘cleavingfunctionality’ must be sufficiently reactive with DNA at 37° C., beinert in aqueous media, and not react with buffer components, and notsuffer unimolecular decomposition in competition with desired reactionson DNA. In order to design such bifunctional molecules, hairpinpolyamides equipped with either an N-terminal bromoacetyl group or aprodrug analogue of the cyclopropyl electrophile of CC-1065 have beenprepared.

A. Bromoacetylated polyamides

The polyamide NH₂PyPyPyPy-g-ImPyPyPy-b-Dp was designed to target thesequence 5′-AgTTT*A-3′. T* indicates the thymine opposite the alkylatedadenine. The polyamide was synthesized by solid phase protocols, cleavedfrom the solid support with dimethyl amino propylamine, and purified byreverse phase HPLC chromatography. The terminal pyrrole residue wasdeprotected and left unacetylated, leaving a free primary amine on theN-terminus.

In order to bromoacetylate the polyamide, bromoacetic acid was activatedwith HOBt and DCC in 1 ml DMF. After 5 minutes, the DCU was filtered offand the solution added to the polyamide with DIEA. After 15 minutes, thereaction mixture was purified directly by reversed phase HPLC to isolatethe bromoacetylated polyamide. Short reaction times were used to avoidalkylation of the unprotected imidazole ring nitrogen. The purifiedN-bromoacetyl hairpin polyamide was characterized by mass spectrometry.The synthesis of a bromoacetylated hairpin polyamide is described inFIG. 19.

Another set of polyamides was synthesized, based on an extended hairpinmotif. This motif combines the γ-turn of the hairpin motif with theβ-alanine spacer of the extended motif, combining the 2:1 binding modewith the 1:1 binding mode. The following compounds were synthesized:PyPy-β-PyPyPy-γ-ImPyPy-β-Dp, PyPyPy-β-PyPyPy-γ-ImPyPy-β-Dp,PyPy-β-PyPyPy-γ-PyPyIm-β-Dp, and PyPyPyPyPyPy-γ-PyPyIm-β-Dp. Thesyntheses of the bromoacetylated extended hairpins were successful, andwere prepared as described for the bromoacetylated hairpin polyamide. Ascontrols, all four of the acetylated compounds were made as well.

B. Typical manual solid phase polyamide synthesis ofPyPyPyPy-γ-ImPyPyPy-β-Dp.

Boc-β-alanine-Pam resin (1.25 g, 0.25 mmol) was placed in a 20 ml glassreaction vessel and shaken in DMF for 5 minutes and drained. The resinwas washed with DCM (2 volumes) and deprotected with 80% TFA/DCM/0.5 MPhSH (1 wash, 1×20 minutes). Following deprotection, the resin waswashed 3 time with DCM and 1 time with DMF. Boc-pyrrole-OBt ester (357mg, 1 mmol) was added in 2 ml of DMF followed by 1 ml DIEA. The couplingreaction was shaken vigorously for 45 minutes. Resin samples (5 mg) weretaken periodically to monitor the synthesis by HPLC. Successive cyclesof the remaining monomers, Boc-Py-OBt (2×), Boc-γ-Im-COOH, Boc-Py-OBt(4×). Boc-γ-Im-COOH was activated by addition of HBTU (378 mg, 1 mmol)in 2 ml of DMF. DIEA (1 ml) was added and the solution was allowed tostand for 5 minutes until clear. After completion of the synthesis, theresin was washed with DMF, DCM, methanol, and ethyl ether. The resin wasthen lyophilized to remove solvent. The polyamide was cleaved off theresin with (N,N)-dimethylamino propylamine (2 ml) in a glassscintillation vial at 55° C. for 12 hours. The polyamide was filteredand HPLC purified in 0.1% TFA with a 0.25% CH₃CN min⁻¹ gradient.

C. Synthesis of bromoacetylated polyamides

Bromoacetic acid (65 mg, 0.5 mmol) and hydroxybenzotriazole (65 mg, 0.5mmol) were dissolved in 1 ml DMF. DCC (102 mg, 0.5 mmol) was added.After 5 minutes, the DCU was filtered off, and the solution added to thepolyamide (10 mg, 0.1 mmol). The filter was washed with 1 ml DMF, and100 μl DIEA which was added to the reaction. The reaction was allowed tosit at room temperature for 15 minutes. HPLC purification in 0.1% (w/v)TFA with gradient elution of 0.25% CH₃CN min⁻¹ Bromoacetylated polyamidewas recovered, (0.184 mg, 135.6 μmol). UV λ_(max)(ε): 312 nm (66,600).MALDI-TOF MS: 1358.3 (1357.29 calculated for M+1).

E. AcPyPyPyPy-γ-ImPyPyPy-Dp

Synthesized as above. UV λ_(max)(ε): 318 nm (66,600). MALDI-TOF MS:1279.5 (1279.4 calculated for M+1).

F. NH₂PyPy-β-PyPyPy-γ-ImPyPy-β-Dp

Synthesized as above. UV λ_(max)(ε): 310 nm (66,600). ¹H NMR (DMSO-d₆) δ10.25 (s, 1H), 9.98 (m, 2H), 9.9 (m, 3H), 9.72 (m, 2H), 9.3 (1H, br s),8.04-8.02 (m, 4H), 7.44 (s, 1H), 7.23 (d, 1H), 7.20 (d, 1H), 7.18 (s,1H), 7.17 (d, 1H), 7.167 (s, 1H), 7.145 (s, 1H), 7.119 (s, 1H), 7.08 (s,1H), 7.025 (s, 1H), 6.9 (s, 1H), 6.85 (s, 1H), 6.80 (d, 1H), 6.79 (s,1H), 3.90 (s, 3H), 3.85 (s, 3H), 3.81 (s, 3H), 3.80 (s, 3H), 3.77 (s,3H), 3.66 (br, 12H), 3.43-3.34 (m, 8H), 3.17 (m, 2H), 3.08 (m, 2H), 2.98(m, 2H), 2.32 (m, 6H), 1.74 (m, 4H).

G. AcPyPy-β-PyPyPy-γ-ImPyPy-β-Dp

To a solution of NH₂PyPy-β-PyPyPy-γ-ImPyPy-β-Dp in DMSO/NMP (500 μl) andDIEA (500 μl) was added acetic anhydride (400 μl). The reaction washeated at 55° C. for 15 minutes and HPLC purified as above. UVλ_(max)(ε): 310 nm (66,600). MALDI-TOF MS: 1351.0 (1350.5 calculated forM+1).

H. BrAcPyPy-β-PyPyPy-γ-ImPyPy-β-Dp

Synthesized as bromoacetylated polyamide above. UV λ_(max)(ε): 314 nm(66,600). MALDI-TOF MS: 1429.3 (1429.4 calculated for M+1).

I. NH₂PyPyPy-β-PyPyPy-γ-ImPyPy-β-Dp

Synthesized as above. UV λ_(max)(ε): 314 nm (74,925). ¹H NMR (DMSO-d₆) δ10.25 (s, 1H), 10.02 (s, 1H), 9.99 (s, 1H), 9.90 (m, 3H), 9.72 (m, 2H),8.03-8.06 (m, 4H), 7.44 (s, 1H), 7.23-7.21 (m, 3H), 7.17-7.12 (m, 4H),7.09 (d, 1H), 6.98-6.83 (m, 6H), 3.92 (s, 3H), 3.87 (s, 3H), 3.82 (s,3H), 3.79 (s, 3H), 3.77 (s, 3H), 3.51 (m, 12H), 2.72 (m, 4H), 2.48 (m,4H), 2.32 (m, 6H) 1.78 (m, 4H).

J. AcPyPyPy-β-PyPyPy-γ-ImPyPy-β-Dp

Acetylated as above. UV λ_(max)(ε): 314 nm (74,295). MALDI-TOF MS:1472.0 (1472.6 calculated for M+1) NH₂PyPy-β-PyPyPy-γ-PyPyIm-β-Dp.Synthesized as above. UV λ_(max)(ε): 310 nm (66,600). ¹H NMR (DMSO-d₆) δ10.31 (s, 1H), 9.98 (s, 1H), 9.91 (s, 1H), 9.89 (s, 1H), 9.84 (s, 1H),9.71 (br, 2H), 8.06-8.08 (m, 3H), 7.95 (s, 1H), 7.48 (s, 1H), 7.28-7.15(m, H), 7.08 (s, 1H), 7.02 (m, 2H), 6.91-6.86 (m, 3H), 6.80 (s, 1H),3.91 (s, 3H), 3.86 (s, 3H), 3.82-3.78 (m, 12H), 3.56-3.43 (m, 12H), 3.22(m, 2H), 3.10 (m, 2H), 3.0 (m, 2H), 2.4 (m, 2H), 2.3 (m, 6H), 1.7 (m,4H)

K. AcPyPy-β-PyPyPy-γ-PyPyIm-β-Dp

Acetylated as above. UV λ_(max)(ε): 310 nm (66,600). MALDI-TOF MS:1350.0 (1350.5 calculated for M+1) NH₂PyPyPyPyPyPy-γ-PyPyIm-β-Dp.Synthesized as above. UV λ_(max)(ε): 310 nm (74,925). ¹H NMR (DMSO-d₆) δ10.31 (s, 1H), 10.04 (s, 1H), 9.95 (d, 3H), 9.90 (s, 1H), 9.84 (d, 2H),8.08-8.06 (m, 3H), 7.48 (s, 1H), 7.28 (s, 1H), 7.23-7.21 (m, 2H), 7.15(s, 1H), 7.10-7.07 (m, 8H), 6.94-6.87 (m, 4H), 3.95-3.78 (m, ), 3.58 (m,2H), 3.43 (m, 2H), 3.19 (m, 2H), 3.07 (m, 2H), 2.36 (m, 2H), 2.32 (m,6H), 2.25 (m, (2H), 1.74 (m, 4H).

L. AcPyPyPyPyPyPy-γ-PyPyIm-β-Dp

Acetylated as above. UV λ_(max)(ε): 310 nm (74,295). MALDI-TOF MS:1401.0 (1401.5 calculated for M+1). BrAcPyPyPyPyPyPy-γ-PyPyIm-β-Dp.Bromoacetylated as above. UV λ_(max)(ε): 310 nm (74.295). MALDI-TOF MS:1480.7 (1480.4 calculated for M+1).

M. Alkylation reactions

Alkylation was examined on a 262 bp restriction fragment (EcoRI/FspI) ofpBR322, radiolabeled on the 3′ end (10,000 cpm/reaction). Polyamide orbromodistamycin were added at appropriate concentrations. Final reactionconcentrations were 10 mM sodium phosphate (pH 7.0), 100 μM sonicatedcalf thymus DNA. The reactions were incubated at 37° C. for 0, 1, 5, 10,20, and 40 hours. Following incubation, the reactions were ethanolprecipitated and dissolved in 10 μl 10 mM sodium phosphate buffer andheated at 90° C. for 15 minutes. Piperidine (40 μl, 1.4 M) was added andthe reaction heated again for 15 minutes at 90° C. Piperidine waslyophilized off and the reactions were resuspended in 7 μl 1× TBE/80%formamide loading buffer, denatured by heating at 85° C. for 10 minutesand placed on ice. Reactions were electrophoresed on 8% polyacrylamidegels (5% cross link, 7 M urea) in 1× TBE at 2000 V. Gels were dried andexposed to a storage phosphor screen (Molecular Dynamics).

N. NH₂PyPyPyPy-γ-ImPyPyPy-NH(CH₂)₂OH

Polyamide was synthesized as above on glycine linked Pam resin. Forcleavage, resin (500 mg) was weighed out into a 50 ml flask in 5 ml 100%EtOH. An equal weight of LiBH₄ (500 mg, 23 mmol) was slowly added. Resinwas incubated at 55° C. for 2 hours, adding more ethanol as needed.Polyamide was HPLC purified as above. UV λ_(max)(ε): 314 nm (66,600).MALDI-TOF MS: 1124.0 (1124.2 calculated). ¹H NMR (DMSO-d₆) δ 10.26 (s,1H), 10.02 (s, 1H), 9.99 (s, 1H), 9.94 (d, 2H), 9.89 (d, 2H), 8.02 (m,1H), 7.91 (m, 1H), 7.43 (s, 1H), 7.24-7.20 (m, 2H), 7.16-7.12 (m, 2H),7.09-7.02 (m, 4H), 6.92-6.84 (m, 4H), 3.92-3.87 (m, 6H), 3.83-3.77 (m,18H).

Example 4 Polyamide CBI Unit

(+)CC-1065 is a natural product isolated from Streptomyces zelensis. Itbinds in the minor groove and shows antitumor activity due to a reactivecyclopropyl moiety which alkylates preferentially at N3 of adenine(Boger and Johnson. Angew. Chem. Int. Ed. Eng. 1996, 35, 1438-1474).

Also in this class of antitumor antibiotics are the duocarmycins. Theyare structurally very similar to CC-1065, having the reactivecyclopropyl ring, but lacking the third conjugated ring system. Thesecompounds bind in AT tracts, and display strong sequence selectivity foralkylation at adenines. Alkylation will occur at N3 of guanine as well,but only when other AT bp are protected in the minor groove. Theflanking sequence preferences for alkylation by CC-1065 are5′-AAA-3′>5′-TTA-3′>5′-TAA-3′>5′-ATA-3′. The alkylation reaction isreversible for the two duocarmycin compounds but irreversible forCC-1065. This discrepancy is explained by the more extensivenon-covalent interactions of CC-1065 with the DNA minor groove.(+)CC-1065 is the natural enantiomer. The unnatural enantiomer has beensynthesized by Boger and coworkers and shown to alkylate DNA as well.Interestingly, where the natural enantiomer binds 3′ to 5′ from the siteof alkylation, the unnatural enantiomer binds 5′ to 3′. Structures of(+) CC-1065 and the duocarmycins are shown in FIG. 20.

When compared to N-Bromoacetyldistamycin, CC-I065 shows very differentreactivity. For reaction times of 1 hour at 37° C.,N-Bromoacetyldistamycin shows almost no visible cleavage, while(+)CC-1065 shows intense cleavage at 13 adenines. After 10 hours at 37°,N-Bromoacetyldistamycin shows a comparable amount of cleavage to (+)CC-1065 at 1 hour, but at only one adenine. Despite the apparentsimilarities between these two molecules, being crescent-shaped with anelectrophile that covalently binds DNA, the cyclopropyl electrophile ofCC-1065 alkylation shows faster kinetics than that ofN-Bromoacetyldistamycin. The alkylation mechanism for CC-1065 is shownin FIG. 21.

Several pro-drug analogues of CC-1065 have also been made. One of themost popular is bizelesin, a bifunctional interstrand DNA crosslinkersynthesized by Upjohn. It is believed to go through the same cyclopropylintermediate as CC-1065, but is more stable than the cyclopropylanalogues. The structures of Bizelesin and CBI are shown in FIG. 22.

Boger et al have synthesized many modified versions of the A ring of(+)CC-1065 to examine the effects of steric changes on the alkylationpotency of these drugs. In his work with CC-1065 derivatives, it hasbeen shown that there is a direct linear correlation between drugstability and cytotoxicity. The more solvolytically stable compoundsalso show the highest degree of cytotoxicity. The most successfulmodification thus far, is the synthesis of1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) which replacesthe fused pyrrole with a fused benzene ring, releasing ring strain inthe system. (Boger, D. L., Yun, W., and Han, N. Bioorganic and MedicinalChemistry 1995, 3, 1429-1453.) When coupled to the B and C rings ofCC-1065, CBI showed greater stability, reactivity, and selectivity than(+)CC-1065 itself. Boger has also shown that a Boc protected CBI unit issufficient for DNA alkylation. Its fast kinetics and efficientalkylation make CBI an ideal candidate to tether to a hairpin togenerate a powerful sequence specific alkylator.

The CBI subunit was synthesized as described by Boger. (Boger, D. L. aMcKie., J. A. J. Org. Chem. 1995, 60, 1271-1275.) Briefly,N-Boc-4-hydroxy-2-napthylamine was synthesized by the condensation ofammonia and 1,3 dihydroxynaphthalene with immediate Boc protection byBoc anhydride. After protection of the alcohol with benzyl bromide,treatment with NIS provided the iodonaphthylamine. Alkylation with allylbromide provided a substrate for a favorable 5-exo-trig arylradical-alkene cyclization to occur, using Bu₃SnH and TEMPO radicaltrap. Cleavage of the TEMPO trap intermediate occurred upon heating withactivated Zn powder. Treatment with PPh₃/CCl₄, followed by hydrolysis ofthe benzyl ether gave the desired product.

In order to work out the coupling conditions of the polyamide-CBI unit,a simple three ring compound, ImPyPy-β-NH(CH₂)₂NH₂ was made. A newactivation strategy used disuccinimidyl glutarate (DSG), a diacidactivated with NHS esters. A β-alanine linker was added to the CBI unitto facilitate completion of the reaction, according to the procedure byLukhtanov, E. A. and coworkers. (Lukhtanov, et al. Nucleic AcidsResearch 1996, 24, 683-687.) After HPLC purification, one major peak wasisolated. This fraction was analyzed by mass spectrometry and NMR andcould be identified as the polyamide-CBI (chloro) conjugate. Thesynthesis of a CBI-polyamide conjugate is shown in FIG. 23.

A. ImPyPy-β-ED

Polyamide was synthesized as above, and cleaved with ethylene diamine.HPLC purification as above. UV λ_(max)(ε): 300 nm (24,975). ¹H NMR(DMSO-d₆) δ 10.47 (s, 1H), 9.91 (s, 1H), 8.06 (m, 1H), 7.67 (m, 1H),7.38 (s, 1H), 7.25 (s, 1H), 7.15 (s, 1H), 7.12 (s, 1H), 7.02 (s, 1H),6.84 (s, 1H), 3.96 (s, 3H), 3.80 (s, 3H), 3.76 (s, 3H), 3.37 (m, 2H),3.35 (m, 2H), 2.83 (m, 2H), 2.46 (m, 2H).

B. ImPyPy-β-ED-succinimide-NHS

ImPyPy-β-ED (10 mg) was dissolved in 2 ml DMF added 100 μl at a time toa solution of disuccinimidyl glutarate (100 mg) and DIEA (10 μl) in 1 mlDMF at room temperature. The reaction was monitored by analytical HPLCand was complete within an hour after final addition of polyamide.Preparative HPLC gave a white powder. MS (FAB): 695.2 (calculated694.3). ¹H NMR (DMSO-d₆) δ 10.49 (s, 1H), 9.91 (s, 1H), 8.01 (m, 1H),7.92 (m, 1H), 7.87 (m, 1H), 7.38 (s, 1H), 7.25 (s, 1H), 7.17 (s, 1H),7.11 (s, 1H), 7.04 (s, 1H), 6.78 (s, 1H), 3.96 (s, 3H), 3.80 (s, 3H),3.76 (s, 3H), 3.33 (m, 2H), 3.05 (m, 2H), 2.77 (m, 2H), 2.65 (m, 2H),2.55 (m, 2H), 2.30 (m, 2H), 2.28 (m, 2H), 2.16 (m, 2H), 2.13 (m, 2H),1.78 (m, 2H).

C. Boc-β-alanine-CBI

Deprotect alcohol (217 mg, 0.65 mmol) as above. After removing ethylacetate, dissolve in dry DMF (10 ml). Add to Boc-β-alanine (245.98 mg,1.3 mmol) and EDC (767 mg, 4 mmol). Reaction was stirred under argonovernight. Solvent was removed in vacuo and precipitated in 20 ml ofwater. The precipitate was centrifuged, washed, with water, andlyophilized. Flash chromatography gave a yellow powder. ¹H NMR (DMSO-d₆)δ 9.43 (br s, 1H), 8.31 (s, 1H), 8.29 (s, 1H), 7.65 (d, 1H), 7.53 (t,1H), 7.40 (t, 1H), 5.56 (m, 1H).

D. CBI conjugated polyamide

Boc-β-alanine CBI was deprotected as above. A solution ofImPyPy-β-ED-succinimide-NHS (20 mg) in 100 μl DMF was added with 10 μlDIEA. Reaction was stirred at room temperature under argon overnight.HPLC purification gave a white powder. MS(FAB): 884.1 (calculated 884.4)¹H NMR (DMSO-d₆) δ 10.50 (s, 1H), 10.36 (br s, 1H), 9.91 (s, 1H), 8.04(m, 2H), 7.92 (m, 2H), 7.87 (m, 1H), 7.81 (m, 1H), 7.73 (m, 2H), 7.44(t, 2H), 7.41 (s, 1H), 7.27-7.25 (m, 3H), 7.16 (s, 1H), 7.10 (s, 1H),7.06 (s, 1H).

Example 5 Polyamide-Intercalator Conjugates

The artificial regulation of protein:DNA interactions is a potentiallypowerful therapeutic tool. Protein recognition of DNA, both specific andnon-specific, is based heavily on the nearby DNA structure. (Luisi, B.(1995) in DNA-Protein: Structural Interactions, ed. Lilley, D. M. J.(IRL Press, Oxford), p. 23.) For example, a bent sequence of DNA mayrecruit a non-specific protein, as in HMG-I, or prevent a protein frommaking the appropriate contacts for high-affinity binding. Smallmolecules designed to bind predetermined sequences of DNA and modulatethe local DNA topology may be a general approach for regulation of thefunction of DNA binding proteins.

Intercalators are a class of molecules which are potent antibiotic andantitumor drugs. (Neidle and Abraham, (1984) CRC Crit. Rev. Biochem. 17,73-121. Wang, A. H-J. (1992) Curr. Opin. Struct. Biol. 2, 361-368.)Lerman first described intercalation as the insertion of a flat,aromatic chromophore between adjacent base pairs of the double helix.(Lerman, L. S. (1961) J. Mol. Biol. 3, 18-30.) The rise of B-form DNA isusually 3.4 A/base pair. The stacking of the intercalator separates theadjacent base pairs by another 3.4 A and extends the length of the helixand equivalent amount per bound intercalator. The base pairs neighboringthe intercalation site are also unwound 10-26° with respect to oneanother. Generally, it is these structural distortions introduced byintercalation which are considered to be the basis for their therapeuticactivity. However, it is important to note that in most cases the DNAhelix returns to its B-form structure within a few base pairs of theintercalation site.

Due to their nature of stacking between the base pairs, intercalatorsgenerally exhibit little or no sequence specificity. A few naturalproducts, such as actinomycin D and the anthracycline and pluramycinfamilies of intercalators, have added functionalitites which impartpreference for certain dinucleotide steps. (Hansen and Hurley (1996)Acc. Chem. Res. 29, 249-\.) Actinomycin D, consists of an aromaticphenoxazone core coupled to two identical cyclic pentapeptides that makecontacts to the exocyclic amine of guanine, granting specificity forintercalation at 5′-GC-3′ steps. Similarly, carbohydrate moietiesattached to the chromophore of the anthracycline and pluramycinintercalators interact with the DNA bases in both the major and minorgrooves and grant these molecules their sequence preferences. In almostall cases, the sequence specificity of these natural products is limitedto the two base pairs adjacent to the intercalation site.

Netropsin and distamycin A are pyrrole carboxamide natural productswhich bind in the minor groove of DNA at sites of 4-5 contiguous A,Tbase pairs. (Krylov, et al. (1979) Nucleic Acids Res. 6, 289-304;Zasedatelev, et al. (1974) Mol. Biol. Rep. 1, 337-342; Zasedatelev, etal. (1976) Dokl. Akad. Nauk SSSR (1976) 231, 1006-1009; Zimmer, andWanhert (1986) Prog. Biophys. Mol. Biol. 47, 31-112; Van Dyke, et al.(1982) Proc. Natl. Acad. Sci., USA 79, 5470-5474; Van Dyke and Dervan(1982) Cold Spring Harbor Symp. Quant. Biol. 47, 347-353; Van Dyke andDervan, (1983) Biochemistry 47, 2373-2377; Harshman and Dervan (1985)Nucleic Acids Res. 13, 4825-4835; Fox and Waring, (1984) Nucleic AcidsRes. 12, 9271-9285; Lane et al. (1983) Proc. Natl. Acad. Sci., USA 80,3260-3264). In an attempt to create an intercalator with designedsequence specificity, a number of researchers have linked analogs ofdistamycin or netropsin to a non-specific intercalator. (Bailly andHenichart, (1991) Bioconj. Chem. 2, 379-393; Bourdouxhe-Housiaux, et al.(1996) Biochemistry 35, 4251-4264; Bailly, et al. (1994) Biochemistry33, 15348-15364; Subra, et al. (1991) Biochemistry 30, 1642-1650;Eliadis, et al. (1988) J. Chem. Soc. Chem. Comm. 1049-1052; Wang, et al.(1994) Gene 149, 63-67; Arcamone, F. (1994) Gene 149, 57-61.) Althoughthese efforts have met with some success, these compounds target mixedsequences of A•T and G•C base pairs. More specifically none of thesecompounds can bind a broad range of predetermined DNA sequences. Evenmore specifically, none of these compounds can bind a predeterminedsequence with subnanomolar affinity.

Linking a non-specific intercalator moiety to a polyamide may producethe sequence specific distortions of DNA structure required to regulateprotein:DNA interactions. Ethidium bromide is a common intercalatorwhich has been shown to bind DNA with a Ka of approximately 10⁵ M⁻¹ andunwind the DNA helix by 26°. (LePecq and Paoletti (1967) J. Mol. Biol.27, 87-106; Waring, M. (1970) J. Mol. Biol. 54, 247-279; Wang, J. C.(1974) J. Mol. Biol. 89, 783-801; Bresloff and Crothers (1975) J. Mol.Biol. 95, 103-123). A derivative of ethidium, methidium, has been usedpreviously in the preparation of designed intercalators and serves asthe basis of methidium-propyl-Fe•EDTA (MPE) footprinting. (Dervan andBecker (1978) J. Am. Chem. Soc. 100, 1968-1970; Hertzberg and Dervan(1982) J. Am. Chem. Soc. 104, 313-315.) The synthesis and DNA-bindingproperties of a series of methidium-polyamide conjugates have beendiscovered by the present inventor.

Methidium-polyamide conjugates are designed to sequence specificallyinduce helical unwinding and extension which may be sufficient toinhibit DNA binding by a wide variety of DNA binding proteins, such asthe transcription factor, GCN-4, SP1, and NF-κB.

A. Design and Synthesis of Methidium-Polyamide Intercalators

A series of methidium-polyamide conjugates of the general designDMγ-ImPyPy-γ-ImPyPy-β-C_(n)-Mdm (DMγ=N,N-dimethyl-γ-aminobutyric acid,C_(n)=diamine linker of n carbons, Mdm=p-carboxymethidium) weresynthesized. Polyamides generally contain a C-terminal positivelycharged dimethylaminopropyl amide. In this case, since the C-terminus isconjugated to the methidium, DMγ was placed on the N-terminus to retainthe net positive charge. This alteration has no significant effect onpolyamide binding. Boc-chemistry solid phase polyamide synthesis allowsfor the rapid preparation of milligram quantities of purified polyamidesuitable for methidium conjugation in solution.DMγ-ImPyPy-γ-ImPyPy-β-Pam-resin was prepared from Boc-Py-OBt ester andBoc-Im acid monomers. Aminolysis with various diamines (NH₂(CH₂)_(n)NH₂, n=2, 4, 6) followed by preparatory HPLC purification affordedfree polyamide with a C-terminal primary amine suitable for coupling tomethidium. Reaction of the polyamide amine with the acylimidazole esterofp-carboxy methidium and HPLC purification produced a series ofmethidium-polyamide conjugates with various linker lengths. Thepolyamide/methidium coupling reaction was quantitative by analyticalHPLC. An average recovery of purified conjugate of 14.5% fromDMγ-ImPyPy-γ-ImPyPy-β-C_(n)-NH₂ was acheived. The ¹H NMR spectrum ofeach conjugate has resonances consistent with polyamide and methidiumprotons, as well as an additional broad triplet at 8.75 ppm resultingfrom the amide bond formed in the polyamide/methidium coupling reaction.MALDI-TOF mass spectrometry analysis of each conjugate reveals thepresence of compounds consistent with the mass of the conjugatedspecies, with no free polyamide or methidium observed. The synthesis ofbifunctional methidium-polyamide conjugates is described in FIG. 24.

DMγ-ImPyPy-γ-ImPyPy-β-C_(n)-Mdm conjugates are targeted to the5′-TGACT-3′ portion of the ARE and GCRE binding sites of GCN4. By CPKmodeling, intercalation is expected to occur between the two base pairsat the 3′ end of the GCN4 biding site, AT for ARE (5′-CTGACTAAT-3′) andTT GCRE (5′-ATGACTCTT-3′) (intercalation site bolded). Coupling of themethidium (K_(a) 10⁵ M⁻¹) and polyamide (K_(a) 10⁵ M⁻¹) moieties is alsoexpected to produce a significant increase in binding affinity. Bindingof a methidium-polyamide conjugate to a 5′-AGTGTA-3′ site is depictedbelow. The methidium is represented as a grey rectangle and is placedbetween the base pairs where intercalation is predicted based onmolecular modeling studies.

¹H NMR were recorded on a GE 300 instrument operating at 300 MHz.Spectra were recorded in DMSO-d₆ with chemical shifts reported in partsper million relative to residual DMSO-d₅. UV spectra were measured on aHewlett-Packard Model 8452A diode array spectrophotometer.Matrix-assisted, laser desorption/ionization time of flight massspectrometry was carried out at the Protein and Peptide MicroanalyticalFacility at the California Institute of Technology. HPLC analysis wasperformed either on a HP 1090 M analytical HPLC or a Beckman Gold systemusing a Rainen C18, Microsorb MV, 5 μm, 300×4.6 mm reversed phase columnin 0.1% (wt/v) TFA with acetonitrile as eluent and a flow rate of 1.0ml/min, gradient elution 1.25% acetonitrile/min. Preparatory HPLC wascarried out on a Beckman instrument using a Waters DeltaPak 25×100 mm100 μm C₁₈ column in 0.1% (wt/v) TFA, gradient elution 0.25%/min. CH₃CN.Wafer was obtained from a Millipore Milli-Q water purification system.Reagent-grade chemicals were used unless otherwise stated. Restrictionendonucleases were purchased from either New England Biolabs orBoeringher-Mannheim and used according to the manufacturer's protocol.Sequenase (version 2.0) was obtained from United States Biochemical, AndDNase I (FPLCpure) was obtained from Boeringher-Mannheim.[α-³²P]-Thymidine-5′-triphosphate (3000 C_(i)/mmol), and[α-³²P]-deoxyadenosine-5′-triphosphate (6000 C_(i)/mmol), were purchasedfrom Du Pont/NEN.

B. Synthesis of Polyamide-Methidium Conjugates

Boc-Im acid and Boc-Py-OBt were synthesized in 5 and 6 steps,respectively. DMγ-ImPyPy-γ-ImPyPy-β-Pam-resin was prepared usingBoc-chemistry manual solid phase synthesis protocols. Polyamide wascleaved from the resin (400 mg) by aminolysis in neat diamine (2 mL,24-48 hours, 60° C.) and purified by preparative HPLC. p-Carboxymethidium acid (50 mg) in DMSO (1 mL) was activated by reaction withcarbonyl diimidazole (22 mg) and N-ethylmorpholine (15 μL) in DMSO (200μL) (25° C., 1 hour). Aliquots of this solution (375 μL) and DIEA (150μL) were added to DMγ-ImPyPy-γ-ImPyPy-β-C_(n)-NH₂ (n=2, 4, 6)polyamides, each in DMSO (150 μL). After 12-24 hours the reaction wasdiluted with 0.1% (wt.v) TFA (5 mL) and purified by HPLC.

C. Dmγ-ImPyPy-γ-ImPyPy-β-C₂-Mdm

Coupling of p-carboxy methidium acid acid toDMγ-ImPyPy-γ-ImPyPy-β-C₂-NH₂ (20 mg, 19 μmol) affordedDMγ-ImPyPy-γ-ImPyPy-β-C₂-Mdm as a purpose powder. (3.0 mg, 2 μmol, 10.5%recovery). HPLC r.t. 28.9, UV λ_(max) (ε), 290 (93,000); ¹H NMR(DMSO-d₆); d 10.38 (s, 1 H), 10.25 (s, 1 H), 9.99 (s, 1 H), 9.95 (s, 1H), 9.89 (s, 2 H), 9.4 (br s, 1 H), 8.80 (t, 1 H), 8.65 (t, 2 H), 8.18(d, 2 H), 8.08 (m, 3 H), 7.73 (d, 2 H), 7.48 (d, 1 H), 7.42 (s, 1 H),7.41 (s, 1 H), 7.30 (d, 2 H), 7.23 (s, 2 H), 7.15 (m, 2 H), 6.86 (s, 1H), 6.82 (s, 1 H), 6.30 (d, 1 H), 3.93 (s, 9 H), 3.82 (s, 3 H), 3.80 (s,3 H), 3.78 (s, 6 H), 3.38 (m, 2 H), 3.18 (m, 2 H), 3.08 (m, 4 H), 2.76(d, 6 H), 2.35 (m, 9 H), 1.92 (m, 2 H), 1.76 (m, 2 H). MALDI-TOF MS1390.8 (1390.6 calc. for M+H).

D. DMγ-ImPyPy-γ-ImPyPy-β-C₄-Mdm

Coupling of p-carboxy methidium acid acid toDMγ-ImPyPy-γ-ImPyPy-β-C₄-NH₂ (35 mg, 32 μmol) affordedDMγ-ImPyPy-γ-ImPyPy-β-C₄-Mdm as a purpose powder. (8.7 mg, 6 μmol, 19%recovery). HPLC r.t. 29.7, UV λ_(max) (ε), 290 (93,000); ¹H NMR(DMSO-d₆); d 10.38 (s, 1 H), 10.25 (s, 1 H), 9.97 (s, 1 H), 9.95 (s, 1H), 9.90 (s, 2 H), 9.4 (br s, 1 H), 8.74 (t, 1 H), 8.61 (t, 2 H), 8.17(d, 2 H), 8.02 (m, 2 H), 7.95 (t, 1 H), 7.73 (d, 2 H), 7.48 (d, 1 H),7.43 (s, 1 H), 7.42 (s, 1 H), 7.30 (d, 2 h), 7.23 (s, 2 H), 7.15 (m, 2H), 6.87 (s, 1 H), 6.82 (s, 1 H), 6.30 (d, 1 H), 3.93 (m, 6 H), 3.92 (s,3 H), 3.82 (s, 3 H), 3.81 (s, 3 H), 3.77 (s, 6 H), 3.38 (m, 2 H), 3.18(m, 2 H), 3.08 (m, 4 H), 2.76 (d, 6 H), 2.40 (m, 9 H), 1.92 (m, 2 H),1.78 (m, 2 H), 1.50 (m, 4 H). MALDI-TOF MS 1418.7 (1418.6 calc. forM+H).

E. DMγ-ImPyPy-γ-ImPyPy-β-C₆-Mdm

Coupling of p-carboxy methidium acid acid toDMγ-ImPyPy-γ-ImPyPy-β-C₆-NH₂ (30 mg, 27 μmol) affordedDMγ-ImPyPy-γ-ImPyPy-β-C₆-Mdm as a purple powder. (5.3 mg, 3.7 μmol, 14%recovery). HPLC r.t. 30.6, UV λ_(max) (ε), 290 (93,000); ¹H NMR(DMSO-d₆); d 10.38 (s, 1 H), 10.26 (s, 1 H), 9.98 (s, 1 H), 9.95 (s, 1H), 9.90 (s, 2 H), 9.4 (br s, 1 H), 8.73 (t, 1 H), 8.61 (t, 2 H), 8.17(d, 2 H), 8.02 (m, 2 H), 7.89 (t, 1 H), 7.73 (d, 2 H), 7.48 (d, 1 H),7.44 (s, 1 H), 7.43 (s, 1 H), 7.30 (d, 2 H), 7.24 (s, 2 H), 7.14 (m, 2H), 6.88 (s, 1 H), 6.83 (s, 1 H), 6.31 (d, 1 H), 3.93 (m, 9 H), 3.83 (m,6 H), 3.78 (s, 6 H), 3.31 (m, 2 H), 3.18 (m, 2 H), 3.03 (m, 4 H), 2.76(d, 6 H), 2.35 (m, 9 H), 1.92 (m, 2 H), 1.78 (m, 2 H), 1.50 (m, 6 H).MALDI-TOF MS 1446.7 (1446.9 calc. for M+H).

Example 6 Polyamide Dye Conjugates

Solution methods for the sequence-specific detection of nucleic acidsoffer several advantages in terms of sample preparation and of timeresolution of measurements. Currently most efforts in this directionfocus on hybridization methods of single stranded targets. The targetingof double helical DNA allow for the direct detection of biological DNAsamples including plasmid, cosmid, or genomic DNA. DNA-bindingpyrrole-imidazole polyamide will sequence-specifically deliverenvironmentally sensitive fluorochromes to the DNA. Several dyes show amarkedly increased fluorescence upon binding to DNA, among these areHoechst 33258, ethidium bromide, and most notably thiazole orange. Moregenerally, dyes such as dansyl and mansyl show tremendous sensitivity toenvironment.

Conjugates have been prepared with a number of such dyes in order todevelop sequence-specific, high affinity DNA fluorochromes. Thepolyamide portion of each dye was prepared using solid phase syntheticmethodology and reacted with an amine reactive fluorochrome. A number ofdyes and ‘linker diamines’ are being investigated. These conjugates areunique, in that they combine the ability to recognize any predeterminedDNA sequence with the ability to signal binding events directly.

The synthesis of a polyamide-rhodamine conjugate is outlined below. [(i)80% TFA/DCM 0.4M PhSH; (ii) BocPy-OBt, DIEA, DMF; (iii) 80% TFA/DCM,0.4M PhSH; (iv) BocPy-OBt, DIEA, DMF; (v) 80% TFA/DCM, 0.4M PhSH; (vi)BocPy-OBt, DIEA, DMF; (vii) 80% TFA/DCM, 0.4M PhSH; (viii)Boc-γ-aminobutyric acid-Im-OBt, HBTU, DIEA, DMF; (ix) 80% TFA/DCM, 0.4MPhSH; (x) BocPy-OBt, DIEA, DMF; (xi) 80% TFA/DCM, 0.4M PhSH; (xii)BocPy-OBt, DIEA, DMF; (xiii) 80% TFA/DCM, 0.4M PhSH; (xiv) BocPy-OBt,DIEA, DMF; (xv) 80% TFA/DCM, 0.4M PhSH; (xvi) Imidazole-2-carboxylicacid (HBTU/DIEA); (xvii) diamino-N-methyldipropylamine, 55° C.; (xviii)5-carboxyrhodamine 6G succinimidyl ester, 20 mM HEPES, pH 7.5, 25° C.](FIG. 25). The chemical structures of a number of polyamide-DYEconjugates are shown in FIGS. 26A-D.

Example 7 DNA Detection Through Energy Transfer

Systems which show enhanced or specific fluorescence upon binding to aspecific DNA sequence could be useful reagents for genomic analysis.Energy transfer between Dyes provides a means of detecting simultaneousbinding of sequence-specific imidazole-pyrrole polyamides to proximalDNA binding sites. (Ju, et al. Proc. Natl. Acad. Sci. 92, 4347-4351; Nieet al., Science 266, 1018-1021). Two DNA binding polyamides will beprepared to target adjacent DNA binding site, one conjugated to a donordye, the other conjugated to an acceptor dye. Dye pairs will be chosensuch that the donor dye can be excited without exciting the acceptor.With excitation at this energy, fluorescence of the acceptorfluorochrome will only occur while proximal to the donor fluorochromethorugh energy transfer from the donor. The required binding of the twopolyamides will lengthen the effective recognition sequence to thelevels appropriate for genomic level analysis and will improve thespecificity of the technique.

Using dye conjugation chemistry developed by the present inventor,conjugates will be prepared purified, and characterized. Donor-acceptorpairs such as fluorescein-rhodamine or thiazole-orange/rhodamine will beanalyzed for their computability in this system. This energy transfersystem increases the currently accessible recognition sequence forpolyamides and provides for a unique binding-dependent signal,applicable for both homogenous and heterogenous detection systems.

Pyrene and similar systems for excimers (excited state dimers) providetwo or more molecules are close in three dimensional space. DNA-bindingpolyamides deliver pyrene to proximal positions on DNA. Binding is thenmonitored by the formation of the excimer.

The structures of a pyrene conjugate is shown below:

Example 8 Polyamide Biotin Conjugates

Conjugates prepared between sequence specific DNA binding polyamides andbiotin are useful for a variety of applications. First, such compoundscan be readily attached to a variety of matrices through the stronginteraction of biotin with the protein streptavidin. (Weber. P. C.,Ohlendorf, D. H., Wendoloski, J. J., Salemme, F. R. Science 243, 85-88)Readily available strepdavidin-derivatized matrices include magneticbeads for separations as well as resins for chromatography. (Ito, T.,Smith, C. L., Cantor, C. R. Proc. Natl. Acad. Sci. 89, 495-498; Tagle,D. A., Swaroop, M., Lovett, M., Collins, F. S. Nature 361, 751-753)

A number of such polyamide-biotin conjugates have been synthesized bysolid phase synthetic methods. Following resin cleavage with a varietyof diamines, the polyamides were reacted with carious biotin carboxylicacid derivatives to yield conjugates. The conjugates were purified byHPLC and characterized by MALDI-TOF mass spectroscopy and 1H NMR. Thesynthesis of biotin-polyamide conjugate is shown in FIG. 27. Thechemical structure of a number of bifunctional biotin-polyamideconjugates prepared by the present invention are shown in FIGS. 28A-D. Ascheme for sequence specific affinity capture by a bifunctionalpolyamide-biotin conjugates is outlined in FIG. 29.

Example 9 Photoactivated Modification of DNA By A Polyamide-PsoralenConjugate

Photoactivated modification of DNA by a polyamide-psoralen conjugate.Psoralen and psoralen derivatives have been used as photoactive drugs inthe treatement of cancer. (Edelson, et al. N. Engl. J. Med. 316, 297(1987)) These molecules intercalate into double-helical DNA and uponirradiation with UVA undergo a [2+2] cycloaddition reaction with the 5,6double bond of thymine residues to form both monoadducts and interstrandDNA cross-links. (Psoralen DNA Photobiology, Volumes 1 and 2; Gesparro,F. P., Ed. CRC Press, Inc., Boca Raton, Fla. 1988.) Our recent interestshave focused on the synthesis and in vitro analysis of photoactivepolyamide-psoralen conjugate B which is desinged to form covalentattachments to DNA in a sequence-specific manner. The use of light as atrigger for the permanent covalent modification of DNA may prove to beattractive tool for potential in vivo applications such as the specificinhibition of transcription by minor groove binding polyamides. Theextended hairpin polyamide-psoralen conjugate B was synthesized bycoupling the OBt ester of S-(8-psoralenyloxy)pentanoic acid. (Lee, etal., J. Med. Chem. 1994, 37, 1208.) to the extended hairpin polyamidedirectly on the β-alanine-Pam resin. Upon equilibration of thepsoralen-polyamide conjugate at pH 7.5 with a 247 bp restrictionfragment followed by irradiation at 360 nm, the extent of intrastrandcross-link formation was shown to be between 15-20% and 54-57% at 10 nMand 100 nM concentrations of polyamide respectively. Our current workinvolves the use of a polymerase stop assay as a tool to map the sitesof intrastrand covalent modification as well as sites of potentialmonoadduct formation on double-helical DNA. The structure of thepsoralen-polyamide conjugate is shown in FIG. 30.

Example 10 In Vitro Assay for Polyamide Binding

An engineered, radiolabeled restriction fragment from pUC-19 wasprepared in which a nine bp polyamide binding site overlaps by two basepairs with the cleavage site for the restriction endonucleases Pvu II.Clevage by Pvu II is prevented when the overlapping polyamide bindingsite is occupied by the polyamide. As a control, a second radiolabeledDNA fragment was prepared which contains a Pvu II site, but lacks theoverlapping polyamide binding site.

The rate of polyamide association with its target binding site wasassessed by combining solutions of the polyamide with the radiolabeledtarget and reference fragments and allowing them to for 5 minutes to 5hours before initiating a treatment (1-2 minutes) with the enzyme PvuII. Under the experimental conditions, the reference site is nearlycompletely digested, but protection at the target site is observed andcan be correlated with polyamide concentration and the time ofequilibration. Similarly, the dissociation rate is analyzed by adding anexcess of unlabeled competitor DNA to an equilibrated solution of thelabeled DNA fragments and polyamide. Addition of the competitor reducesthe concentration of free polyamide to zero. The rate at with polyamidedissociation occurs from the target site on the labeled fragment can befollowed by the rate of loss of protection from Pvu II digestion as there-equilibration time is increased. The association profile with respectto time for the 9-ring extended hairpin polyamideImPyPy-γ-ImPyPy-β-PyPyPy-G-Dp binding its cognate 9 base pair match siteis shown in FIG. 6.

The extended hairpin dissociation rate has been determined to bek_(d)=3.1±0.2×10⁻⁵ s⁻¹, this corresponds to a half-time of 6.2 h (10 mMBis-Tris (pH 7.0), 50 mM NaCl, 5 mM MgCl₂, 1 mM mercaptoethanol at 37°C.). Wherein half-time is defined as the time required for 50% of apopulation of DNA and polyamide to dissociate or associate. Theassociation rate has been determined at k_(a)=1.3±0.8×10⁴ M⁻¹s⁻¹, thiscorresponds to a half-time of 3.0 h at 5.0 nM. The determined value forthe equilibrium association constant (K_(eq)=6.3±0.8×10⁸ M⁻¹) correlateswell with the kinetically determined ratio (k_(a)/k_(d)=4.2±2.6×10⁸M⁻¹).

These results demonstrate that polyamides bind to a designated targetsite within seconds to minutes, but that it may take hours fordissociation to occur at such a site. More specifically these resultsdemonstrate that polyamides bind DNA with a combination of associationand dissociation rates which provide effective modulation of theactivity of DNA binding proteins.

Example 11 Cooperative Hairpin Dimers for Recognition of DNA by Py-ImPolyamides

Small molecules which permeate cells and bind predetermined DNAsequences have the potential to control the expressoin of specificgenes. Trauger, et al. Nature 1996, 382, 559-561; Gottesfeld, et al.Nature 1997, 387, 202-205). Recently, an eight-ring polyamide whichbinds to a six base pair target site was shown to inhibit genetranscription in cell culture (Gottesfeld, et al. Nature 1997, 387,202-205). Polyamides recognizing longer DNA sequences should providemore specific biological activity (P. B. Dervan, Science 1986, 232, 464)which could be achieved by synthesizing larger hairpins (Turner, et al.EJ. Am. Chem. Soc. 1997, 119, 7636-7644). However, the upper limit ofpolyamide size with regard to efficient cell permeation is not known.

Alternatively, a more biomimetic approach is to bind larger DNAsequences while maintaining the size of the polyamide. Nature'stranscription factors often bind large DNA sequence by formation ofcooperative protein dimers at adjacent half-sites (Ptashne, et al. AGenetic Switch, Blackwell Scientific Publications and Cell Press: PaloAlto, Calif. 1986; Pabo, et al. Ann. Rev. Biochem. 1992, 61, 1053-1095;Marmorstein, et al. Nature 1992, 356, 408-414; Klemm, et al. Cell 1994,77, 21-32; Bellon, et al. Nature Struct. Biol. 1997, 4, 586-591). Forcooperatively binding extended Py-Im polyamide dimers, the two ligandscan slip sideways with respect to one another, allowing recognition ofother sequences (Trauger, et al. J. Am. Chem. Soc. 1996, 118, 6160-6166;Swalley , et al. Chem. Eur. J. 1997, 3, 1600-1607). Hairpin polyamidesutilizing the turn-specific γ-aminobutyric acid linker are constrainedto be fully overlapped and preclude the “slipped motif” option (Mrksich,et al. J. Am. Chem. Soc. 1994, 116, 7983-7988; Parks, et al. ibid. 1996,118, 6153-6159; Swalley, et al. ibid. 1996, 118, 8198-8206; Swalley, etal. ibid. 1997, 119, 6953-6961; Trauger, et al. Chem. & Biol. 1996, 3,369-377; Declairac, et al., J. Am. Chem. Soc. 1997, 119, 7909-7916).Provided herein is a cooperative six-ring extended hairpin polyamidewhich dimerizes to specifically bind a predetermined ten base pairsequence.

A sequence contained in the regulatory region of the HIV-1 genome wasselected as the target site (Jones, et al. Ann. Rev. Biochem. 1994, 63,717-743; Frech, et al. Virology 1996, 224, 256-267). To design theligand, the polyamide ring pairing rules provided herein, such as theinclusion of β-alanine (β) to relax ligand curvature, and the preferenceof γ-aminobutyric acid (γ) for a “hairpin turn” conformation withinpolyamide-DNA complexes were considered. This analysis suggested thatthe six-ring polyamide having the core sequence ImPy-β-ImPy-γ-ImPy mightbind the target sequence 5′-AGCAGCTGCT-3′ through formation of acooperative hairpin dimer (FIG. 31). To avoid a collision between theN-terminal end of one ligand and the C-terminal end of the second withinthe complex, the positively-charged β-alanine-dimethylaminoproplyamideC-terminus used in standard polyamides has been replaced with theshorter, uncharged (CH₂)₂OH group (C₂-OH). The cationic “turn” residue(R)-2,4-diaminobutyric acid ((R)^(H2N)γ) maintains the overall +1 chargefor optimal solubility in water.

Polyamide ImPy-β-ImPy-(R)^(H2N)γ-ImPy-C₂-OH was synthesized usingsolid-phase methods (E. E. Baird, P. B. Dervan, J. Am. Chem. Soc. 1996,118, 6141-6146) on glycine-PAM resin (available in 0.3 mmol/gsubstitution from Peptides International, Louisville, Ky.), reductivelycleaved from the solid support using LiBH₄ (Mitchell, et al., J. Org.Chem. 1978, 43, 2845; Stewart, et al. Solid Phase Peptide Synthesis,Pierce Chemical Company, Rockford, Ill., 1984) and purified by HPLC(reverse-phase). The identity and purity of the polyamides was confirmedby ¹H NMR, analytical HPLC, and MALDI-TOF MS. MALDI-TOF MS(monoisotopic) (M+H): ImPy-β-ImPy-(R)^(H2N)γ-ImPy-C₂-OH, obsd 953.3,calcd (C₄₂H₅₃N₁₈O₉) 953.4; ImPy-β-ImPyPy-(R)^(H2N)γ-PyImPy-C₂-OH, obsd1197.5, calcd (C₅₄H₆₅N₂₂O₁₁) 1197.5.

FIG. 32 illustrates the(ImPy-β-ImPy-(R)^(H2N)γ-ImPy-C₂-OH)₂•5′-AGCAGCTGCT-3′ complex,demonstrating binding models for complexes of a 10 base pair match andsingle-base pair mismatch sites (the mismatched base pair is highlightedby shading). The shaded and open circles represent imidazole and pyrrolerings, respectively, diamonds represent β-alanine, half-circlesrepresent (CH₂)₂OH groups, and curved lines represent(R)-2,4-diaminobutyric acid.

FIG. 33a represents a storage phosphor autoradiogram of the 8%denaturing polyacrylamide gel used to separate the fragments generatedby DNase I digestion in a quantitative footprint titration experimentwith polyamide ImPy-β-ImPy-(R)^(H2N)γ-ImPy-C₂-OH: lane 1, A lane; lane2, DNase I digestion products obtained in the absence of polyamide;lanes 3-12, DNase I digestion products obtained in the presence of 0.1,0.2, 0.5, 1, 2, 5, 10, 20, 50, and 100 nM polyamideImPy-β-ImPy-(R)^(H2N)γ-ImPy-C₂-OH, respectively. All reactions containpJT-LTR 3′-³²P-end-labeled EcoRi/HindIII restriction fragment (15 kcpm),10 mM Tris•HCl, 10 mM KCl, 10 mM MgCl₂, and 5 mM CaCl₂ (pH 7.0, 24° C.).Plasmid pJT-LTR was prepared by ligating an insert having the sequence5′-CCGGTAACCAGAGAGACCCAGTACAGGCAAAAAGCAGCTGCTTATATGCAGCATCTGAGGGACGCCACTCCCCAGTCCCGCCCAGGCCACGCCTCCCTGGAAACTCCCCAGCGGAAAGTCCCTTGTAGAAAGCTCGATGTCAGCAGTCTTTGTAGTACTCCGGATGCAGCTCTCGGGCCACGTGATGAAATGCTAGGCGGCTGTCAA TCGA-3′ tothe large AvaI/SalI fragment of pUC19.

Quantitative DNase I footprinting on a 245 base pair 3′-³²P-end-labeledrestriction fragment showed that ImPy-β-ImPy-(R)^(H2N)γ-ImPy-C₂-OH bindsits match stie 5′-AGCAGCTGCT-3′ at nanomolar concentrations (apparentmonomeric association constant, K_(a)=1.9 (±0.3)×10⁸ M⁻¹), and alsobinds a single-base pair mismatch site 5′-AGATGCTGCA-3′ with 9-foldlower affinity, K_(a)=2.2 (±0.5)×10⁷ M⁻¹ (FIG. 33b).

The binding data for match and single-base pair mismatch sites werewell-fit by cooperative binding isotherms, consistent with formation ofcooperative 2:1 polyamide-DNA complexes.⁽⁵⁾ A double-base pair mismatchsite, 5′-AGCTGCATCC-3′, is also bound with 65-fold lower affinity. Thefact that this mismatch site, which contains the “half-site”5′-AGCTGCA-3′, is not effectively bound indicates that recognition ofthe match site occurs through cooperative dimerization, and not due toformation of 1:1 hairpin complexes.

Further study of the generality and sequence specificity of this motifis in progress and will be reported in due course. For example, we foundthat the eight-ring polyamide ImPy-β-ImPyPy-(R)^(H2N)γ-PyImPy-C₂-OHbinds the twelve base pair match site 5′-AAGCAGCTGCTT-3′ with 10-foldhigher affinity than ImPy-β-ImPy-(R)^(H2N)γ-ImPy-C₂-OH, and isapproximately 100-fold specific for this site versus the double-basepair mismatch site 5′-CAGATGCTGCAT-3′.

The DNA-binding affinity and specificity for the six-ring polyamideImPy-β-ImPy-(R)^(H2N)γ-ImPy-C₂-OH for its ten base pair binding site aretypical of standard six-ring hairpins which recognize five base pairs.Thus, use of a the cooperative hairpin dimer motif doubles the bindingsite size relative to the standard hairpin motif without sacrificingaffinity or specificity, and without increasing the molecular weight ofthe ligand. As provided herein, a novel cooperative hairpin dimer motif,relatively low molecular weight pyrrole-imidazole polyamides (MWapproximately 950-1,200) can specifically recognize 10-12 base pairs ofDNA.

FIG. 34 provides general polyamide motifs for use in designingpolyamides having improved binding and specificity. FIG. 35 providesfive general formulas for polyamides of the present invention. FIG. 36illustrates the DNA footprint analysis and affinities of additionalcooperatively-bound polyamides. FIG. 37 demonstrates the N-terminalextension of the polyamide ImPyPy-X-ImImPy-γ-PyPyPy-β-Dp where X is γ,C5-8, β-β, or β-C5.

Table 5 illustrates recognition of 15 Base-Pairs byImPyPy-X-ImImPy-γ-PyPyPy-β-Dp polyamides. Association constants (K_(a))for the match site 5′-AACCAAGTCTTGGTA-3′ and specificities for the matchsite versus center (5′-AACCAACTGTTGGTA-3′) and edge(5′-AACCAAGTCTTGCGA-3′) mismatch sites are also illustrated

TABLE 5 Length of X K_(a) (M⁻¹) Specificity X = (# atoms) Match Centermismatch Edge mismatch γ 5 1 × 10⁷ 1 1 C5 6 4 × 10⁸ 11 19 C6 7 2 × 10⁸12 15 C7 8 2 × 10⁸ 2 2 β—β 8 3 × 10⁸ 2 2 C8 9 2 × 10⁸ 10 10 β-C5 10 1 ×10⁸ 2 2 Solution conditions: 10 mM Tris.HCl, 10 mM KCl, 10 mM MgCl₂, and5 mM CaCl₂ at 24° C. and pH 7.0. The parent hairpinAc-ImImPy-γ-PyPyPy-β-Dp binds both 5 base pair binding sites within the15 base pair target site 5′-AACCAAGTCTTGGTA-3′ with K_(a) = 10⁷ M⁻¹.

Table 6 illustrates recognition of 15 Base-Pairs byImImPy-γ-PyPyPy-X-ImPyPy-β-Dp polyamides. Association constants (K₁) forthe match site 5′-AACCAAGTCTTGGTA-3′ and specificities for the matchsite versus center (5′-AACCAACTGTTGGTA-3′) and edge(5′-AACCAAGTCTTGCGA-3′) mismatch sites are shown.

TABLE 6 Length of X K_(a) (M⁻¹) Specificity X = (# atoms) Match Centermismatch Edge mismatch C5 6 1 × 10⁹ 2 2 C6 7 2 × 10⁸ 1 2 C7 8 1 × 10⁹ 11 β—β 8  1 × 10¹⁰ 1 1 Solution conditions: 10 mM Tris.HCl, 10 mM KCl, 10mM MgCl₂, and 5 mM CaCl₂ at 24° C. and pH 7.0. The parent hairpinImImPy-γ-PyPyPy-β-Dp binds both 5 base pair binding sites within the 15base pair target site 5′-AACCAAGTCTTGGTA-3′ with K_(a) = 10⁷ M⁻¹.

Solution conditions: 10 mM Tris.HCl, 10 mM KCl, 10 mM MgCl₂, and 5 mMCaCl₂ at 24° C. and pH 7.0. The parent hairpin ImImPy-γ-PyPyPy-β-Dpbinds both 5 base pair binding sites within the 15 base pair target site5′-AACCAAGTCTTGGTA-3′ with K_(a)=1×10⁸ M⁻¹.

Table 7 illustrates recognition of 15 base pairs ofImPyPy-X-ImImPy-γ-PyPyPy-C3-OH. Association constants (K_(a)) for thematch site 5′-AACCAAGTCTTGGTA-3′ and specificities for the match siteversus center (5′-AACCAACTGTTGGTA-3′) and edge (5′-AACCAAGTCTTGCGA-3′)mismatch sites.

TABLE 7 K_(a) Specificity Polyamide 5′-AACCAAGTCTTGGTA-3′5′-AACCAACTGTTGGTA-3′ 5′-AACCAAGTCTTGCGA-3′ImPyPy-C5-ImImPy-γ-PyPyPy-C3-OH 3 × 10⁷ >3  >3 ImPyPy-C6-ImImPy-γ-PyPyPy-C3-OH 3 × 10⁷ 2 2ImPyPy-β-β-ImImPy-γ-PyPyPy-C3-OH 1 × 10⁸ 3 3 Ac-ImImPy-γ-PyPyPy-C3-OH 7× 10⁶ — —

Table 8 illustrates recognition of 16 Base-Pairs byImPyPyPy-X-ImImPy-γ-PyPyPy-β-Dp. Association constants (K_(a)) for thematch site 5′-AACCAAGTACTTGGTA-3′ and specificities for the match siteversus center (5′-AACCAACTAGTTGGTA-3′) and edge (5′-AACCAAGTACTTGCGA-3′)mismatch sites.

TABLE 8 K_(a) Specificity Polyamide 5′-AACCAAGTACTTGGTA5′-AACCAACTAGTTGGTA 5′-AACCAAGTACTTG CG AImPyPyPy-C5-ImImPy-γ-PyPyPy-β-Dp 7 × 10⁸ >3  >3 ImPyPyPy-C6-ImImPy-γ-PyPyPy-β-Dp 1 × 10⁸ 1 1ImpyPyPy-β-β-ImImPy-γ-PyPyPy-β-Dp 3 × 10⁸ 1 1 Ac-ImImPy-γ-PyPyPy-β-Dp 3× 10⁷ — —

Table 9 illustrates recognition of recognition of 9-11 base pairs byImPyPy-X-ImPyPy-β-Dp.

TABLE 9 Polyamide 5′-TGTCAGACA-3′ 5′-TGTCAAGACA-3′ 5′-TGTCAAAGACA-3′5′-gcggtTGTCAacccg-3′     ImPyPy-G-ImPyPy-β-Dp 1 × 10⁸ 3 × 10⁷ 3 × 10⁷ 3× 10⁷     ImPyPy-β-ImPyPy-β-Dp 2 × 10⁸ 2 × 10⁸ 1 × 10⁹ 1 × 10⁹    ImPyPy-γ-ImPyPy-β-Dp 5 × 10⁷ 1 × 10⁸ 1 × 10⁸ 2 × 10⁸   ImPyPy-C5-ImPyPy-β-Dp 2 × 10⁸ 1 × 10⁹ 1 × 10⁹ 1 × 10⁹   ImPyPy-C6-ImPyPy-β-Dp 1 × 10⁸ 2 × 10⁸ 5 × 10⁷ 5 × 10⁷   ImPyPy-C7-ImPyPy-β-Dp 2 × 10⁷ 1 × 10⁸ 3 × 10⁷ 7 × 10⁷   ImPyPy-C8-ImPyPy-β-Dp 1 × 10⁷ 3 × 10⁷ 1 × 10⁷ 1 × 10⁷  ImPyPy-C11-ImPyPy-β-Dp 5 × 10⁶ 2 × 10⁷ 2 × 10⁷ 1 × 10⁷   ImPyPy-β-ImPyPy-β-Dp 2 × 10⁸ 2 × 10⁸ 1 × 10⁹ 1 × 10⁹  ImPyPy-β-β-ImPyPy-β-Dp 1 × 10⁷ 5 × 10⁷ 1 × 10⁷ 2 × 10⁷ ImPyPy-β-β-β-ImPyPy-β-Dp 1 × 10⁷ 1 × 10⁷ 7 × 10⁷ 1 × 10⁷ImPyPy-β-Py-β-ImPyPy-β-Dp 3 × 10⁸ 2 × 10⁸ 1 × 10⁹ <10⁸ImPyPy-β-ImPyPy-β-Dp 2 × 10⁸ 2 × 10⁸ 1 × 10⁹ 1 × 10⁹ImPyPy-β-PyPyPy-β-Dp 2 × 10⁸ 5 × 10⁷ 5 × 10⁷ 3 × 10⁷ Solutionconditions: 10 mM Tris•HCl, 10 mM KCl, 10 mM MgCl₂, and 5 mM CaCl₂ and5mM CaCl₂ at 22 °C and pH 7.0.

Table 10 illustrates recognition of 16 Base-Pairs by a polyamide havingthe formula ImPyPyPy-X-ImImPy-γ-PyPyPy-C3-OH. Association constants(K_(a)) for the match site 5′-AACCAAGTACTTGGTA-3′ and specificities forthe match site versus center (5′-AACCAACTAGTTGGTA-3′) and edge(5′-AACCAAGTACTTGCGA-3′) mismatch sites.

TABLE 10 K_(a) Specificity Polyamide 5′-AACCAAGTACTTGGTA 5′-AACCAA C TAG TTGGTA 5′-AACCAAGTACTTGCGA ImpyPyPy-C6-ImImPy-γ-PyPyPy-C3-OH <10⁸ — —Ac-ImImPy-γ-PyPyPy-C3-OH 7 × 10⁶ — —

Table 11 illustrates recognition of 17 Base-Pairs by a polyamide of theformula ImPyPy-β-X-β-ImImPy-γ-PyPyPy-β-Dp, X=β or Py. Associationconstants (K_(a)) for the match site 5′-AACCATAGTCTATGGTA-3′ andspecificities for the match site versus center (5′-AACCATAGCGTATGGTA-3′)and edge (5′-AACCAAGTCTTGCGA-3′) mismatch sites.

TABLE 11 K_(a) Specificity Polyamide 5′-AACCATAGTCTATGGTA5′-AACCATAGCGTATGGTA 5′-AACCATAGTCTATGCGAImPyPy-β-β-β-ImImPy-γ-PyPyPy-β-Dp ? ? ?ImPyPy-β-Py-β-ImImPy-γ-PyPyPy-β-Dp ? ? ? Ac-ImImPy-γ-PyPyPy-β-Dp 3 × 10⁷— —

Example 12 Recognition of 16 Base Pairs in the Minor Groove of DNA by anIm-Py Polyamide Dimer

Cell-permeable small molecules which bind predetermined DNA sequenceswith affinity and specificity comparable to natural DNA-binding proteinshave the potential to regulate the expression of specific genes.Recently, an 8-ring hairpin Py-Im polyamide which binds 6 base pairs ofDNA was shown to inhibit transcription of a specific gene in cellculture (Gottesfeld, et al. Nature 1997, 387, 202-205). Polyamidesrecognizing longer DNA sequences should provide more specific biologicalactivity. To specify a single site within the 3 billion base pair humangenome, ligands which specifically recognize 15-16 base pairs arenecessary. For this reason, recognition of 16 base pairs represents amilestone in the development of chemical approaches to DNA recognition(Dervan, P. B. Science 1986, 232, 464; Dervan, P. B. In The Robert A.Welch Foundation Conference on Chemical Research XXXI. Design of Enzymesand Enzyme Models; Houston, Tex., Nov. 2-4, 1987; pp 93-109; Dervan, P.B. In Nucleic Acids and Molecular Biology, Vol. 2; Springer-Verlag:Heidelberg, 1988; pp 49-64; Moser, et al. Science 1987, 238, 645-650; LeDoan, et al. Nucleic Acids Res. 1987, 15, 7749; Strobel, et al. Science1991, 254, 1639-1642; Thuong , et al. Angew. Chem. Int. Ed. Engl. 1993,32, 666-690). A Py-Im polyamide dimer which targets 16 contiguous basepairs in the minor groove of DNA is provided herein.

As the length of a polyamide dimer having the general sequence ImPy₂₋₆increases beyond 5 rings (corresponding to a 7 base pair binding site),the DNA-binding affinity ceases to increase with polyamide length(Kelly, et al. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6981-6985). Astructural basis for this observation is provided by the recentlydetermined X-ray crystal structure structure of a 4-ring homodimer incomplex with DNA, which reveals a perfect match of polyamiderise-per-residue with the pitch of the DNA duplex, but overwound ligandcurvature (Keilkopf, et al. Nature Struct. Biol. 1998, 5, 104-109). Thecurvature mismatch explains the observation that flexible β-alanineresidues reset an optimum fit of polyamide dimers with the DNA helix atlong binding sites (Trauger, et al. J. Am. Chem. Soc. 1996, 118,6160-6166; Swalley, et al. Chem. Eur. J. 1997, 3, 1600-1607).

The 16 base pair sequence 5′-ATAAGCAGCTGCTTTT-3′ present in theregulatory region of the HIV-1 genome was utilized as a binding site(Jones, et al. Ann. Rev. Biochem. 1994, 63, 717-743; Frech, et al.Virology 1996, 224, 256-267). Consideration of the previously publishedpolyamide ring pairing rules (Wade, et al. J. Am. Chem. Soc. 1992, 114,8783-8794; Mrksich, et al. Proc. Natl. Acad. Sci. 1993, 32, 11385-11389;Geierstanger, et al. J. Am. Chem. Soc. 1993, 115, 4474-4482; White, etal. Chem. & Biol. 1997, 4, 569-578; Pelton, et al. Proc. Natl. Acad.Sci. U.S.A. 1989, 86, 5723-5727; Pelton, et al. J. Am. Chem. Soc. 1990,112, 1393-1399; Chen, et al. Nature Struct. Biol. 1994, 1, 169-175;White, et al. Biochemistry 1996, 35, 12532-12537), the A, T specificityof β/β pairs, and the “slipped” dimer motif (Geierstanger, et al. NatureStruct. Biol. 1996, 3, 321-324; Trouger, et al. Chem & Biol. 1996, 3,369-377) suggested that the 8-ring polyamideImPy-β-ImPy-β-ImPy-β-PyPy-β-Dp (1) would specifically bind the targetsequence as a cooperative antiparallel dimer (FIG. 38).

FIG. 38 illustrates a model of the complex ofImPy-β-ImPy-β-ImPy-β-PyPy-β-Dp (1, R=H) orImPy-β-ImPy-β-ImPy-β-PyPy-β-Dp-EDTA.Fe(II) (1-E, R=R_(E))Im=N-methylimidazole, Py=N-methylpyrrole, β=β-alanine, Dp=dimethylaminopropylamide) with 5′-ATAAGCAGCTGCTTTT-3′. The shaded andopen circles represent imidazole and pyrrole rings, respectively, andthe diamonds represents β-alanine. Circles with dots represent lonepairs on N3 of purines and O2 of pyrimidines, and circles containing anH represent the N2 hydrogen of guanine. Putative hydrogen bonds areillustrated by dashed lines. The polyamides were synthesized usingsolid-phase methods (Baird, et al. J. Am. Chem. Soc. 1996, 118,6141-6146), purified by HPLC, and the identity and purity confirmed by¹H NMR, analytical HPLC and MALDI-TOF MS.

A quantitative DNase I footprinting experiment carried out on a 245 basepair 3′-³²P-end-labeled restriction fragment revealed that the polyamidespecifically binds it target site at subnanomolar concentrations(apparent monomeric association constant, K_(a) 3.5×10¹⁰ M⁻¹) (FIG. 39)(Baird, et al. J. Am. Chem. Soc. 1996, 118, 6141-6146; Brenowitz, et al.Methods Enzymol. 1986, 130, 132-181; Cantor, C. R.; Schimmel, P. R.,Biophysical Chemistry, Part III: The Behavior of BiologicalMacromolecules; W. H. Freeman, New York, N.Y., 1980, p 863).

FIG. 39 illustrates a storage phosphor autoradiogram of an 8% denaturingpolyacrylamide gel used to separate the fragments generated by DNAse Idigestions in a quantitative footprint titration experiment withpolyamide ImPy-β-ImPy-β-ImPy-β-PyPy-β-Dp (1): lane 1, A lane; lane 2,DNase I digestion products obtained in the absence of polyamide; lanes3-10, DNase I digestion products obtained in the presence of 0.01, 0.02,0.05, 0.1, 0.2, 0.5, and 1 nM polyamide ImPy-β-ImPy-β-ImPy-β-PyPy-β-Dp,respectively; lane 11, intact DNA. All reactions contain3′-³²P-end-labeled EcoRI/HindIII restriction fragment from plasmidpJT-LTR (15 kcpm), 10 mM Tris.HCl, 10 mM KCl, 10 mM MgCl₂, and 5 mMCaCl₂ (pH 7.0, 24° C.). b) Autoradiogram of a gel used to separate thefragments generated by an affinity cleavage reaction using polyamideImPy-β-ImPy-β-ImPy-β-PyPy-β-Dp-EDTA.Fe (II) (1-E). Lanes 1 and 5: Asequencing lanes; lanes 2-4: cleavage products obtained in the presenceof 0.03, 0.1, 0.3 and 1 nM ImPy-β-ImPy-β-ImPy-β-PyPy-β- Dp-EDTA.Fe (II),respectively; lane 6: intact DNA. All reactions contain labeledrestriction fragment (7 kcpm), and 20 mM HEPES, 300 mM NaCl, 50 μg/mLglycogen, 1 μM Fe (II), and 5 mM DTT (pH 7.3, 24° C.). The sequence ofthe restriction fragment in the region of the 16 base pair target siteand a model of the (ImPy-β-ImPy-β-ImPy-β-PyPy-β-Dp-EDTA.Fe (II)) ₂.DNAcomplex are shown along the right side of the autoradiogram. Lineheights are proportional to the observed cleavage intensity at theindicated base.

The method used for determining association constants involves theassumption that [L]_(tot)˜[L]_(free), where [L]_(free) is theconcentration of polyamide free in solution (unbound). For very highassociation constants this assumption becomes invalid, resulting inunderestimated association constants. In the experiments described here,the DNA concentration is estimated to be ˜5 pM. As a consequence,apparent association constants greater than 1-2×10¹⁰ M⁻¹ represent alower limit of the true association constant. The binding data werewell-fit by a cooperative binding isotherm, consistent with formation ofa cooperative 2:1 complex. To provide further evidence thatImPy-β-ImPy-β-ImPy-β-PyPy-β-Dp binds as an extended dimer, an affinitycleavage experiment was carried out with the polyamide-EDTA.Fe (II)conjugate of ImPy-β-ImPy-β-ImPy-β-PyPy-β-Dp shown in FIG. 39b. Cleavagewas observed at each end of the match sequence, consistent with adimeric, antiparallel binding mode. With regard to sequence specificity,there is a proximal two-base pair mismatch site, 5′-cAGATGCTGCATATa-3′,to the 5′ side of the ³²P-labeled strand which is bound with at least35-fold lower affinity than the match site. However, other mismatchsites on the restriction fragment are bound with 10-20-fold loweraffinity, revealing limitations of this first effort at 16 base pairrecognition. Undoubtedly there is ample room for further optimization ofsequence specificity.

The high binding affinity and the affinity cleavage pattern observed forthe 16 base pair polyamide.DNA complex indicates that 8 pairs of amideresidues form a fully overlapped core which properly positions the 6Im/Py pairs for recognition of 6 G,C base pairs and 2 β/β pairs forrecognition of 2 A,T base pairs. Polyamides composed of 2-ring subunitsconnected by β-alanine appear to be isohelical with B-DNA, and allowplacement of Imidazole residues at any ring position, thus providing ageneralizable motif for recognition of predetermined DNA sequences. Thedata presented herein allows for the design of polyamides capable ofbinding 16 base pairs of DNA at subnanomolar concentrations of suitablesize for permeating cells (i.e., MW˜1,200).

The references described throughout this specification are fullyincorporated by reference. While a preferred form of the invention hasbeen shown in the drawings and described, since variations in thepreferred form will be apparent to those skilled in the art, theinvention should not be construed as limited to the specific form shownand described, but instead is as set forth in the claims.

I claim:
 1. A polyamide of the formula

or a pharmaceutically acceptable salt thereof, wherein: n is 5-11; m is0-6; each Y is independently selected from the group consisting of

wherein p is 0 to 5, provided that at least one Y is not —NH-; Z is acovalent bond or

wherein r is 1-3; each X is independently selected from the groupconsisting C and N; each R₁ is independently selected from the groupconsisting of H, C₁₋₆ alkylamine, C₁₋₆ alkyldiamine, C₁₋₆alkylcarboxylate, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl and C₁₋₆alkyl-L; each R₂ independently selected from the group consisting of Hand OH if X is C, and is not present if X is N; R₃ is selected from thegroup consisting of H, Cl, NO, N-acetyl, benzyl, C₁₋₆ alkyl, C₁₋₆alkenyl, and C₁₋₆ alkynyl; and R₄ is —NR⁵R⁶ or —NH(CH₂)₀₋₆NR₅R₆, whereR₅ and R₆ are independently chosen from the group consisting of H, Cl,NO, N-acetyl, benzyl, C₁₋₆ alkyl, C₁₋₆ alkylamine, C₁₋₆ alkyldiamine,C₁₋₆ alkylcarboxylate, C₁₋₆ alkenyl, C₁₋₆ alkyl, and C₁₋₆ alkyl-L;wherein L is a detectable label, and wherein at least one R₁, R₅, of R₆is C₁₋₆ alkyl-L.
 2. The polyamide of claim 1, wherein one or more R₂ isH, one or more R₁ is CH₃, and one or more X is N.
 3. The polyamide ofclaim 1 selected from the group consisting of ImPyPyPy-γ-PyPyPyPy-β-Dp,PyPyImPy-γ-PyPyPyPy-β-Dp, ImPyPyPy-γ-ImPyPyPy-β-Dp,PyImPyPy-γ-PyImPyPy-β-Dp, ImPyImPy-γ-PyPyPyPy-β-Dp,ImImPyPy-γ-PyPyPyPyβ-Dp, ImImImPy-γ-PyPyPyPy-β-Dp,ImImPyPy-γ-ImPyPyPy-β-Dp, ImPyPyPy-γ-ImImPyPy-β-Dp,ImImPyPy-γ-ImImPyPy-β-Dp, ImPyImPy-γ-ImPyImPy-β-Dp,ImImImPy-γ-ImPyPyPyPy-β-Dp, and ImImImIm-γ-PyPyPyPy-β-Dp, bound to adetectable label, wherein Im is N-methyl-Imidazole, Py is methylpyrrole,βis β-alanine, Dp is dimethylaminopropylamide, and γis γ-aminobutyricacid.
 4. The polyamide of claim 1, wherein said polyamide binds to adouble-stranded DNA molecule with an association constant of at least10⁸ M⁻¹.
 5. The polyamide of claim 4, wherein said polyamide binds to adouble-stranded DNA molecule with an association constant of at least10⁹ M⁻¹.
 6. The polyamide of claim 1, wherein the detectable label is afluorescent molecule.
 7. The polyamide of claim 1, wherein thedetectable label is biotin.
 8. A polyamide selected from the groupconsisting of ImPyPyPy-γ-PyPyPyPy-β-Dp, PyPyImPy-γ-PyPyPyPy-β-Dp,ImPyPyPy-γ-ImPyPyPy-β-Dp, PyImPyPy-γ-PyImPyPy-β-Dp,ImPyImPy-γ-PyPyPyPy-β-Dp, ImImPyPy-γ-PyPyPyPy-β-Dp,ImImImPy-γ-PyPyPyPy-β-Dp, ImImPyPy-γ-ImPyPyPy-β-Dp,ImPyPyPy-γ-ImImPyPy-β-Dp, ImImPyPy-γ-ImImPyPy-β-Dp,ImPyImPy-γ-ImPyImPy-β-Dp, ImImImPy-γ-ImPyPyPyPy-β-Dp,ImImImIm-γ-PyPyPyPy-β-Dp, Im-β-PyPy-γ-Im-β-PyPy-β-Dp,Im-β-ImIm-γ-Py-β-PyPy-β-Dp, Im-β-ImPy-γ-Im-β-ImPy-β-Dp,ImPyPyPyPy-γ-ImPyPyPyPy-β-Dp, ImImPyPyPy-γ-ImPyPyPyPy-β-Dp,ImPyImPyPy-γ-ImPyPyPyPy-β-Dp, ImImPyImIm-γ-PyPyPyPyPy-β-Dp,ImPyPyImPy-γ-ImPyPyImPy-β-Dp, ImPy-β-PyPy-γ-ImPy-β-PyPy-β-Dp,ImIm-β-ImIm-γ-PyPy-β-PyPy-β-Dp, ImPy-β-ImPy-γ-ImPy-β-ImPy-β-Dp,ImPy-β-PyPyPy-γ-ImPyPy-β-PyPy-β-Dp, ImIm-β-PyPyPy-γ-PyPyPy-β-PyPy-β-Dp,ImPy-β-ImPyPy-γ-ImPyPy-β-PyPy-β-Dp, ImIm-β-PyPyPy-γ-ImImPy-β-PyPy-β-Dp,ImPy-β-PyPyPy-γ-PyPyPy-β-ImPy-β-Dp, ImPyPyPyPyPy-γ-ImPyPyPyPyPy-β-Dp,ImPyPy-β-PyPy-γ-ImPyPy-β-PyPy-β-Dp, ImPyPyPy-β-Py-γ-Im-β-PyPyPyPy-β-Dp,ImImPyPyPyPy-γ-ImImPyPyPyPy-β-Dp, Im-β-PyPyPyPy-γ-Im-β-PyPyPyPy-β-Dp,ImPyPyPy-β-Py-γ-ImPyPyPy-β-Py-β-Dp, ImPyImPyPyPy-γ-ImPyPyPyPyPy-β-Dp,ImPyPy-β-PyPy-γ-ImPy-β-PyPyPy-β-Dp, ImPyPyPyPy-β-γ-ImPyPyPyPy-β-β-Dp,ImPy-β-ImPyPy-γ-ImPy-β-ImPyPy-β-Dp, Im-β-PyPyPyPy-γ-ImPyPyPy-β-Py-β-Dp,Im-β-ImPyPyPy-γ-ImPyPyPy-β-Py-β-Dp, ImPyPy-β-PyPyPy-β-Dp,ImImPy-β-PyPyPy-β-Dp, ImImIm-β-PyPyPy-β-Dp, ImPyPyPyPy-β-PyPyPy-β-Dp,ImPyPyPy-β-PyPyPy-β-Dp, ImPyPy-β-PyPyPyPyPy-β-Dp,ImPyPyPy-β-PyPyPy-β-Dp, ImImPyPy-β-PyPyPyPy-β-Dp,ImImImPy-β-PyPyPyPy-β-Dp, ImPyPyPy-β-ImPyPyPy-β-Dp,ImImPyPy-β-ImPyPyPy-β-Dp, ImImPyPyPy-β-PyPyPyPyPy-β-Dp,ImImImPyPy-β-PyPyPyPyPy-β-Dp, ImIm-β-PyPy-β-PyPy-β-PyPy-β-Dp,ImImPy-β-PyPyPy-β-PyPyPy-β-PyPyPy-β-Dp,PyImPy-γ-ImPyPy-β-PyPyPy-β-PyPyPy-β-Dp,PyImPy-γ-ImPyPy-β-PyPyPy-β0PyPyPy-β-Dp, ImImPy-γ-ImPyPy-β-PyPyPy-β-Dp,ImPyPy-γ-ImPyPy-G-PyPyPy-β-Dp, ImPyPyPy-γ-ImImImPy-β-PyPyPy-β-Dp,ImImPyPy-β-Dp, ImImPyPy-γ-ImImPyPy-β-PyPyPyPy-β-Dp, andImImPyPy-γ-PyPyPyPy-β-PyPyPyPy-β-Dp, bound to a detectable label whereinIm is N-methyl-Imidazole, Py is methylpyrrole, βis β-alanine, Dp isdimethylaminopropylamide, and γ is γ-aminobutyric acid.